Treatment of skin conditions by dickkopf1 (dkk1)

ABSTRACT

The present disclosure is generally related to methods of inducing non-palmoplantar skin to develop a palmoplantar phenotype, for example, methods for increasing skin thickness, decreasing skin pigmentation, and/or decreasing hair growth. In particular, disclosed herein are methods of using topical administration of DKK1 to increase skin thickness, decrease skin pigmentation, or reduce hair growth. Also disclosed are topical DKK1 compositions for inducing non-palmoplantar skin to develop a palmoplantar phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 12/518,052, filed onJun. 5, 2009, issued as U.S. Pat. No. 8,198,244, which is a U.S.National Stage of International Application No. PCT/US2007/086855, filedDec. 7, 2007, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Application No.60/873,874 filed Dec. 7, 2006. Each of these applications isincorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The present disclosure is generally related to methods of inducingnon-palmoplantar skin to develop a palmoplantar phenotype, for example,methods for increasing skin thickness, decreasing skin pigmentation,and/or decreasing hair growth.

BACKGROUND

There are a number of topographical, anatomical, and site-specificdifferences between human skin on the sole and the palm (also calledpalmoplantar skin or palmoplantaris) and on the trunk (also callednon-palmoplantar skin or non-palmoplantaris) in terms of thickness andpigmentation. Not only is palmoplantar skin much thicker and less hairythan is skin in other regions of the body, but melanocytes in thoseareas are less dense and produce significantly less melanin pigment thannon-palmoplantar skin.

Many dermatological conditions would be improved by an increase in skinthickness, a decrease in skin pigmentation, or a decrease in hairgrowth. For instance, skin thickening would be desirable in a subjectthat has a skin graft, a skin ulcer, a skin abrasion oravulsion/excision (such as one that leaves a volume defect), an injuryor predisposition to injury caused by a repetitive impact or mechanicalstress, age-related skin changes (for instance, thinning or wrinkledskin), or skin damage due to steroid treatment. A decrease in skinpigmentation would be helpful when the subject has a condition such asuneven skin pigmentation, hyperpigmentation, post-inflammatorypigmentation, ephelides, fragrance dermatitis, sun-damaged skin, apigmented birthmark, lentigos, lichen simplex chronicus, melasma,porphyria cutanea tarda, Addison's disease, Peutz-Jeghers syndrome,acanthosis nigricans, or when depigmentation is desired in a subjectwith widespread vitiligo. Decreased hair growth may be cosmeticallydesirable on an upper or lower extremity or axillary skin, or when asubject has, for instance, hirsutism, congenital adrenal hyperplasia,polycystic ovarian syndrome, hypertrichosis, porphyria cutanea tarda, orincreased vellus hair due to anorexia nervosa.

Given the foregoing, it would be desirable to have methods orcompositions that could induce non-palmoplantar skin to adopt one ormore of the characteristics of palmoplantar skin.

SUMMARY OF THE DISCLOSURE

Disclosed herein is a method for inducing non-palmoplantar skin (forinstance, skin of the trunk, face, or a non-palmoplantar extremity suchas a finger or toe) to develop a palmoplantar phenotype (for instance,becoming one or more or all of thicker, less pigmented, and less hairy).The method includes topically applying an effective amount of DKK1 tonon-palmoplantar skin, which induces the non-palmoplantar skin todevelop a palmoplantar phenotype. In some embodiments, the method is amethod of increasing skin thickness, whereas in other embodiments, themethod is a method of reducing skin pigmentation. In still otherembodiments, the method is a method of reducing hair growth, and instill other embodiments, the method is a method of treating orpreventing melanoma. Also disclosed are pharmaceutical compositions thatinclude an amount of DKK1 sufficient to induce non-palmoplantar skin todevelop a palmoplantar phenotype and a carrier for topicaladministration.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of aseveral embodiments which proceeds with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of digital images showing DKK1 expression bypalmoplantar and by non-palmoplantar fibroblasts. FIG. 1A is a pair ofdigital images of immunoblot gels. The intensity of the 35 kDa DKK1 bandis higher in palmoplantar fibroblasts (Palmi1 Palm2) than innon-palmoplantar fibroblasts (Trunk1, Trunk2). FIG. 1B is a pair ofdigital images showing an immunocytochemical analysis; high levels ofDKK1 are seen in palmoplantar fibroblasts (Palm3) compared withnon-palmoplantar fibroblasts (Trunk3). Nuclei are stained with DAPI.

FIG. 2 is a set of digital images of gels validating gene expressionlevels using RT-PCR in normal human melanocytes untreated (control) ortreated with 50 ng/ml rhDKK1 for two hours. The expression of PKCβ1(p=<0.0001), Krn1(p=<0.0001), LRP6 (p=0.065), LDLR (p=<0.001), GPR51(p=<0.01), TNFR10 (p=<0.0001) and Gadd45 (p=<0.002) is up-regulated inmelanocytes when treated with rhDKK1, but MITF (p=<0.001) isdown-regulated. GAPDH expression served as control (p=0.41) (n>=3).

FIG. 3 is a set of digital images of gels validating expression levelsof proteins related to Wnt signaling pathways in normal humanmelanocytes untreated (control) or treated with 50 ng/ml rhDKK1 for twohours (FIG. 3A) or five days (FIG. 3B) by immunoblotting. Numbers underthe gels indicate levels of intensity compared with β-actin.

FIG. 4 is a set of digital images showing the expression of pGSK3β, PKCαand β-catenin by normal human melanocytes untreated (control) or treatedwith 50 ng/ml rh DKK1 for two hours by immunocytochemistry. Theexpression of GSK3β phosphorylated at Ser9 and PKCα is up-regulated inresponse to rhDKK1, but β-catenin expression is down-regulated.Fluorescence marks antibody staining, nuclei are stained with DAPI.

FIG. 5 is a set of digital images showing immunostaining for β-catenin,PKCα, GSKβ and Ser9-phosphorylated GSKβ in the reconstructed epidermisafter ten days of treatment with or without 100 ng/ml rhDKK1. Note thedecreased expression of β-catenin and GSK3β and the increased expressionof PKCα and Ser9-phosphorylated GSK3β.

FIG. 6 is a set of digital images showing immunohistochemistry for GSKβ,PKCα and β-catenin in palmoplantar and non-palmoplantar skin in situ.Melanocytes in the skin are identified by MART-1 staining. Palm skin(left) shows low expression of GSK3β but high expression of GSK3βphosphorylated at Ser9 and of PKCα. In contrast, levels of β-cateninexpression are lower in palmoplantar melanocytes in situ compared withmelanocytes in trunk skin (right).

FIG. 7 is a set of digital images of cell cultures showing melaninuptake and proliferation of mock-transfected or DKK1-transfectedkeratinocytes in vitro. DKK1-transfected keratinocytes showed lessmelanin phagocytosis than mock-transfected controls (top, bright fieldmicroscopy), whereas the cell density was increased in DKK1-transfectedcells (bottom, phase contrast microscopy). Cell numbers show the numberof cells/field in a counting chamber while melanin content reflectsanalysis by ScionImage, as detailed below in Example 6. Numbers reportedin all figures are means±SEM, and statistical analyses were performedusing Student's t test (NS=not significant).

FIG. 8 is a set of digital images of gels showing mRNA levels in humankeratinocytes treated (+) or untreated (−) with 50 ng/ml rhDKK1 for twohours as analyzed by RT-PCR. The expression of KLEIP and GJB6 wasup-regulated by DKK1, but MITF, P4HA2, Tulp3 and PAR2 weredown-regulated; densitometric analyses are reported at the bottom rightof the figure. GAPDH is shown as a loading control.

FIG. 9 is a set of digital images of gels showing expression patterns ofproteins expressed by keratinocytes three days after transfection withDKK1 (+) or a mock vector (−) and analyzed by Western blot. Expressionof αKLEIP (arrow), PAR2 (no specific band recognized), and otherproteins as noted are shown. Nuclear and cytoplasmic expression ofβ-catenin is shown, as is the cytoplasmic expression of PKCα, PKClβ2,total ERK and phosphorylated ERK (pERK), total GSK3β and phosphorylatedGSK3β (pGSK3β). DKK1 expression was only seen in DKK1-transfectedkeratinocytes, as expected. β-actin is shown as a loading control.

FIG. 10 is a set of digital images of immunocytochemistry ofkeratinocytes treated with (+) or without (−) rhDKK1 for three days.FIG. 10A shows that β-catenin (top) expression was down-regulated byDKK1. The expression of PKCα (middle) and PKCβ1 (bottom) wasup-regulated by DKK1. β-catenin was stained in all panels; yellowindicates colocalization. Nuclei are identified by DAPI. FIG. 10B showsstaining patterns of GSK3β (top), pGSK3β (middle) and pERK (bottom) inDKK1-treated keratinocytes. β-catenin and DAPI staining as for FIG. 10A.

FIG. 11 is a series of digital images showing confocal microscopy of Wntsignaling proteins in keratinocytes treated with (+) or without (−)rhDKK1 for three days. β-catenin (top) expression was down-regulatedwhile pGSK3β (top) and PKCα (bottom) were up-regulated in response toDKK1.

FIG. 12 is a series of digital images showing immunohistochemistry ofsole, palm and dorsal skins. The expression of PAR2 and of β-catenin wasdecreased in palms and soles as compared to dorsal skin. In contrast,keratin 9 was observed only in palmoplantar skin.

FIG. 13 is a series of digital images showing treatment of MelanoDermskin reconstructs with DKK1. FIG. 13A shows the visual aspect of thereconstructed epidermis after 10 days of treatment with or withoutrhDKK1. The epidermis treated with rhDDK1 appeared thicker and lesspigmented than the control. FIG. 13B shows Fontana-Masson staining after4, 7 and 10 days of treatment with or without rhDKK1. The untreatedepidermis showed more melanin than that treated by rhDKK1. FIG. 13Cshows Hematoxylin-eosin staining after 4, 7 and 10 days of treatmentwith or without rhDKK1. The DKK1-treated skin had a thicker stratumcorneum than controls.

FIG. 14 is a series of digital images showing immunohistochemistry ofMelanoDerm reconstructed epidermis. After 10 days of treatment with orwithout rhDKK1, the expression of PAR2 and β-catenin was down-regulatedalthough some cells positive for keratin 9 were observed.

SEQUENCES

The nucleic sequences listed herein and/or in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

SEQ ID NO: 1 is a human PKC_(β1) sense primer

SEQ ID NO: 2 is a PKC_(β1) antisense primer

SEQ ID NO: 3 is a human Krn1 sense primer

SEQ ID NO: 4 is a Krn1 antisense primer

SEQ ID NO: 5 is a human LRP6 sense primer

SEQ ID NO: 6 is an LRP6 antisense primer

SEQ ID NO: 7 is a human LDLR sense primer

SEQ ID NO: 8 is an LDLR antisense primer

SEQ ID NO: 9 is a human GPR51 sense primer

SEQ ID NO: 10 is a GPR51 antisense primer

SEQ ID NO: 11 is a human TNFRSF10A sense primer

SEQ ID NO: 12 is a TNFSF10A antisense primer

SEQ ID NO: 13 is a human Gadd4513 sense primer

SEQ ID NO: 14 is a Gadd4513 antisense primer

SEQ ID NO: 15 is a MITF sense primer

SEQ ID NO: 16 is a MITF antisense primer

SEQ ID NO: 17 is a GAPDH sense primer

SEQ ID NO: 18 is a GAPDH antisense primer 5′-tccaccaccctgttgctgta-3′

SEQ ID NO: 19 is a human KLEIP sense primer

SEQ ID NO: 20 is a KLEIP antisense primer

SEQ ID NO: 21 is a human GJB6 sense primer

SEQ ID NO: 22 is a GJB6 antisense primer

SEQ ID NO: 23 is a human Snrpn sense primer

SEQ ID NO: 24 is a Snrpn antisense primer

SEQ ID NO: 25 is a human BMP21K sense primer

SEQ ID NO: 26 is a BMP21K antisense primer

SEQ ID NO: 27 is a human P4HA2 sense primer

SEQ ID NO: 28 is a P4HA2 antisense primer

SEQ ID NO: 29 is a human Tulp3 sense primer

SEQ ID NO: 30 is a Tulp3 antisense primer

SEQ ID NO: 31 is a human PAR2 sense primer

SEQ ID NO: 32 is a PAR2 antisense primer

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Overview of SeveralEmbodiments

The methods disclosed herein are based on the surprising finding thattopically-applied DKK1 causes non-palmoplantar skin (for instance, skinof the trunk, head, arms, or legs) to take on the characteristics ofpalmoplantar skin (for instance, becoming more like the skin of thepalms and soles, which is thicker, less pigmented, and less hairy).Thus, the disclosed methods are methods for inducing non-palmoplantarskin to develop a palmoplantar phenotype, and they include topicallyapplying an effective amount of DKK1 to non-palmoplantar skin, whichinduces the skin changes. In some embodiments, the method is a method oftreating a dermatological pathology, for instance a skin graft, a skinulcer, a skin abrasion or avulsion/excision (such as one that leaves avolume defect), an injury or predisposition to injury caused by arepetitive impact or mechanical stress, age-related skin changes (forexample photo-aged skin or thin skin), skin damage due to steroidtreatment, uneven skin pigmentation, hyperpigmentation,post-inflammatory pigmentation, ephelides, fragrance dermatitis,vitiligo (for instance, where depigmentation is desired in a subjectwith widespread vitiligo), a pigmented birthmark (for example a café aulait spot), lentigos, or skin changes due to lichen simplex chronicus,melasma, porphyria cutanea tarda, Addison's disease, Peutz-Jegherssyndrome, acanthosis nigricans, hirsutism, congenital adrenalhyperplasia, polycystic ovarian syndrome, hypertrichosis, or anorexianervosa.

Other embodiments are methods for increasing skin thickness. Someexamples of these methods are carried out in vitro, whereas otherexamples of these methods include topically applying the DKK1 to an areaof skin in need of thickening on a subject. In certain in vivo examples,the subject has a skin graft, an ulcer (for instance, a foot ulcer,particularly a diabetic foot ulcer), an abrasion, an injury caused by arepetitive impact or mechanical stress, age-related skin changes, orskin damage due to steroid treatment.

Further embodiments are methods for reducing skin pigmentation. Someexamples of these methods are carried out in vitro, whereas in otherexamples of the methods the DKK1 is topically applied to an area of skinon a subject, for example a pigmented area where reduced pigmentation isdesired. In certain examples, the subject has uneven skin pigmentation,hyperpigmentation, post-inflammatory pigmentation, ephelides, fragrancedermatitis, sun-damaged skin, vitiligo (for instance, wheredepigmentation is desired in a subject with widespread vitiligo), apigmented birthmark (particularly those characterized by the depositionof excess melanin), lentigos, lichen simplex chronicus, melasma,Addison's disease, Peutz-Jeghers syndrome, porphyria cutanea tarda, oracanthosis nigricans.

Still other embodiments are methods for reducing hair growth. In someexamples, DKK1 is applied topically to an area of skin on a subjectwhere hair growth is to be suppressed, and in particular examples, thearea of skin is on an upper or lower extremity or is axillary skin. Inother examples, the subject has hirsutism, congenital adrenalhyperplasia, polycystic ovarian syndrome, hypertrichosis, porphyriacutanea tarda, or anorexia nervosa. In other embodiments the hair growthis to be reduced for cosmetic purposes.

Yet other embodiments are methods for treating or preventing melanoma.In some examples, the DKK1 is topically applied to an area of skin on asubject, such as an area that contains a hyperplastic or premalignantlegion such as a nevus.

Also disclosed are pharmaceutical compositions for carrying out themethods described above. These compositions include an amount of DKK1sufficient to induce non-palmoplantar skin to develop a palmoplantarphenotype and a carrier for topical administration. In some examples,the composition is a cream, an ointment, a lotion, or a tincture, forinstance one including a liposome or a nanoformulation for enhanceddelivery of DKK1. The compositions are for use in treating theconditions disclosed herein.

II. Abbreviations

BCA: bicinchoninic acid

BMP: bone morphogenic protein

BSA: bovine serum albumin

DKK1: dickkopf 1

Gadd: growth arrest and DNA-damage-inducible gene

GSK: glucose-synthase kinase

GSK3β: glycogen synthase kinase 3β

HMGS: Human Melanocyte Growth Supplement

HOX: homeotic

HPS: Hermansky-Pudlak syndrome

Krn: Kremen

LDLR: low-density lipoprotein receptor

LRP: lipoprotein receptor-related protein

LRP6: lipoprotein receptor-related protein 6

MGM: melanocyte growth medium

MITF: microphthalmia-associated transcription factor

MLPH: melanophilin

MRP: mitochondrial ribosomal protein

PAR2: proteinase-activated receptor-2

PBS: phosphate-buffered saline

PKC: protein kinase C

rhDKK1: human recombinant DKK1

SDS: sodium dodecyl sulfate

Shh: Sonic hedgehog

STX5A: syntaxin 5a

SV2B: synaptic vesicle glycoprotein 2b

TNFRSF10A: tumor necrosis factor receptor superfamily, member 10a

III. Explanation of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanation of terms is provided:

Acanthosis nigricans: a brown to black, poorly defined, velvetyhyperpigmentation of the skin, usually present in the posterior andlateral folds of the neck, the axilla, groin, umbilicus, and otherareas. Acanthosis nigricans occurs due to insulin spillover (fromexcessive production due to obesity or insulin insensitivity) into theskin which results in abnormal growth. The most common cause is insulinresistance, usually from type 2 diabetes mellitus. Other causes arefamilial, obesity, drug-induced, malignant (gastric cancer), andpolycystic ovary syndrome.

Addison's disease: (also known as chronic adrenal insufficiency, orhypocortisolism), Addison's disease is a rare endocrine disorder whichresults in the body not producing sufficient amounts of certain adrenalhormones. Addison's disease refers specifically to primary adrenalinsufficiency, in which the adrenal glands themselves malfunction;secondary adrenal insufficiency occurs when the anterior pituitary glanddoes not produce enough adrenocorticotropic hormone to adequatelystimulate the adrenal glands.

The symptoms of Addison's disease are caused by the failure of theadrenal glands, seated above the kidneys, to produce enough of thehormone cortisol and, in some cases, the hormone aldosterone. Often theproduction of adrenaline is also diminished or eliminated. Addison'sdisease usually develops slowly (over several months), and symptoms maynot present or be noticed until some stressful illness or situationoccurs. Common symptoms are chronic fatigue that gradually worsens,muscle weakness, weight loss and loss of appetite, nausea, diarrhea, orvomiting, low blood pressure that falls further when standing(orthostatic hypotension), and areas of hyperpigmentation known asmelasma suprarenale, which are caused by increases inpro-opiomelanocortin, hyponatraemia due to loss of production of thehormone aldosterone, hyperkalaemia due to loss of production of thehormone aldosterone, and axillary or pubic hypotrichosis.

Age-related skin changes: As people grow older, fine lines and wrinklesbegin to develop. The skin loses its firmness and elasticity. Expressionlines form on the face, and patches of discoloration and areas ofdilated blood vessels appear. These changes occur gradually over time,and can first appear in the second and third decades of life. Suchchanges increase with advancing age, and generally people in theirthird, fourth, fifth, or subsequent decades of life experience at leastsome age-related skin changes.

In addition, photoaging describes aging caused by exposure to the sun'srays. Photoaging, as defined herein, is included in the category ofage-related skin changes, and the amount of photoaging that developsdepends on a person's skin color and their history of long-term orintense sun exposure. People with fair skin who have a history of sunexposure develop more signs of photoaging than those with dark skin. Inthe darkest skin, the signs of photoaging are usually limited to finewrinkles and a mottled complexion.

Photoaging occurs over a period of years. With repeated exposure to thesun, the skin loses the ability to repair itself, and the damageaccumulates. Repeated ultraviolet light exposure breaks down collagenand impairs the synthesis of new collagen and elastin, causing the skinto become loose, wrinkled, and leathery much earlier than would occur inthe absence of sun exposure.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects. Therefore, the general term “subject” isunderstood to include all animals, including, but not limited to,humans, or veterinary subjects, such as other primates, dogs, cats,horses, and cows.

Anorexia nervosa: is a psychiatric diagnosis that describes an eatingdisorder characterized by low body weight and body image distortion.Individuals with anorexia often control body weight by voluntarystarvation, purging, vomiting, excessive exercise, or other weightcontrol measures, such as diet pills or diuretic drugs. It primarilyaffects young adolescent girls in the Western world and has one of thehighest mortality rates of any psychiatric condition, with approximately10% of people diagnosed with the condition eventually dying due torelated factors. Anorexia nervosa is a complex condition, involvingpsychological, neurobiological, and sociological components.

People with anorexia typically have a disturbed electrolyte imbalance,low levels of essential hormones (including sex hormones), chronicallyincreased cortisol levels, and osteoporosis develops as a result ofanorexia in 38-50% of cases, as poor nutrition lead to the retardedgrowth of essential bone structure and low bone mineral density.Hypertrichosis is another common symptom of anorexia.

Congenital adrenal hyperplasia: refers to any of several autosomalrecessive diseases resulting from defects in steps of the synthesis ofcortisol from cholesterol by the adrenal glands. Most of these diseasesinvolve excessive or deficient production of sex steroids and can alteror impair development of primary or secondary sex characteristics inaffected infants, children, and adults.

Examples of problems caused by various forms of congenital adrenalhyperplasia include ambiguous genitalia, vomiting leading to dehydrationand death in early infancy, early development of pubic hair, rapidgrowth in childhood, precocious puberty or failure of puberty to occur,excessive facial hair, virilization, hyperpigmentation, and/or menstrualirregularity in adolescence, and infertility due to anovulation.

Dermatological pathology: is an abnormal condition of the skin,including, but not limited to a condition causing uneven skinpigmentation, hyperpigmentation, unwanted hair growth, or thinning ofthe skin. Specific, nonlimiting examples of dermatological pathologiesinclude a skin graft, an ulcer, an abrasion, an injury caused by arepetitive impact or mechanical stress, age-related skin changes, skindamage due to steroid treatment, uneven skin pigmentation,hyperpigmentation, post-inflammatory pigmentation, fragrance dermatitis,vitiligo, a pigmented birthmark, lentigos, or skin changes due to lichensimplex chronicus, melasma, porphyria cutanea tarda, Addison's disease,Peutz-Jeghers syndrome, acanthosis nigricans, hirsutism, congenitaladrenal hyperplasia, polycystic ovarian syndrome, hypertrichosis, oranorexia nervosa.

DKK-1: a secreted antagonist of canonical Wnt signaling that interactswith Wnt receptor lipoprotein receptor-related protein 6 (LRP6) and withthe transmembrane proteins Kremen (Krn) 1 and 2. DKK1 blocks canonicalWnt signaling by inducing endocytosis of the LRP6 complex withoutaffecting the Wnt receptor Frizzled. DKK1 induces the formation ofectopic heads in Xenopus laevis in the presence of BMP inhibitors, andplays critical roles in modulating apoptosis during vertebrate limbdevelopment (especially inter-digit space formation) by interacting withBMP.

As used herein, the term DKK1 includes both human and non-human DKK1proteins (for example, rat, mouse, and chicken DKK1), as well asfunctional DKK1 fragments. Specific, nonlimiting examples of Dkk-1protein sequences are listed as GenBank Accession Nos. gi37183128,gi31542557, gi7110719, gi13124053, gi4545252, gi10281590, gi118092551,gi62858825, gi115313025, gi13124044, gi114630593, gi114630591,gi114630589, gi29504796, gi16306720, gi46394862, and gi5360731.Specific, non-limiting examples of DKK1 fragments can be found in U.S.Pat. No. 7,057,017. One of ordinary skill in the art will recognize thatthese DKK1 full-length proteins and DKK1 fragments are provided merelyas examples; other proteins that fall into the described class will berecognized.

DNA (deoxyribonucleic acid): a long chain polymer which comprises thegenetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal. The term codon is also used for the corresponding(and complementary) sequences of three nucleotides in the mRNA intowhich the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Thus, areference to the nucleic acid molecule that encodes DKK1 or a fragmentthereof encompasses both the sense strand and its reverse complement.Thus, for instance, it is appropriate to generate probes or primers fromthe reverse complement sequence of the disclosed nucleic acid molecules.

Encode: a polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Ephelides: (freckles) are tanned macules found on the skin. They areusually multiple in number. With sun exposure, they become moreapparent; therefore, in the winter months, they are often imperceptible.Although ephelides are predominantly benign, they may be seen inassociation with systemic disease.

Functional fragments and variants of a polypeptide: included are thosefragments and variants that maintain at least one function of the parentpolypeptide. It is recognized that the gene or cDNA encoding apolypeptide can be considerably mutated or truncated without materiallyaltering one or more of the polypeptide's functions. Functionalfragments and variants can be of varying length. For example, a fragmentmay consist of 10 or more, 25 or more, 50 or more, 75 or more, 100 ormore, or 200 or more amino acid residues, so long as they maintain atleast one function of the parent polypeptide.

Hirsutism: is defined as excessive and increased hair growth inlocations where the occurrence of terminal hair normally is minimal orabsent. It refers to a male pattern of body hair (androgenic hair) andit is therefore primarily of cosmetic and psychological concern.Hirsutism is a symptom rather than a disease and may be a sign of a moreserious medical indication, especially if it develops well afterpuberty.

The cause of hirsutism can be either an increased level of androgens(male hormones) or an oversensitivity of hair follicles to androgens.Male hormones such as testosterone stimulate hair growth, increase sizeand intensify the pigmentation of hair. Other symptoms associated with ahigh level of male hormones include acne, irregular menstrual periods,deepening of the voice, and increased muscle mass.

Hyperpigmentation: the darkening of an area of skin or nails caused byincreased melanin. Hyperpigmentation may be caused by sun damage,inflammation from acne, or other skin injuries, such as cuts, abrasions,burns, and puncture wounds, as well as the resulting scars. It is alsoassociated with a number of diseases or conditions, including Addison'sdisease and other sources of adrenal insufficiency, in which hormonesthat stimulate melanin synthesis are frequently elevated, acanthosisnigricans, or hyperpigmentation of intertriginous areas associated withinsulin resistance, or patchy hyperpigmentation often found in pregnantwomen, linea nigra, a hyperpigmented line found on the abdomen duringpregnancy, post-inflammatory pigmentation, fragrance dermatitis, andPeutz-Jeghers syndrome, an autosomal dominant disorder characterized byhyperpigmented macules on the lips and oral mucosa and gastrointestinalpolyps.

Hypertrichosis: refers to a condition of excessive body hair. It can begeneralized, symmetrically affecting most of the torso and limbs, orlocalized, affecting an area of skin. It may be mild or severe. In mostcases, the term is used to refer to an above-average amount of normalbody hair that is unwanted and is an aspect of human variability. Inmedical practice, once generalized hypertrichosis has been distinguishedfrom hirsutism, it is most often considered a variation of normal,primarily resulting from genetic factors.

Nearly all the skin of the human body (except palms of hands and solesof feet) is covered with hair. The density of the hairs (in hairfollicles per square centimeter), thickness of the hairs, color of thehairs, speed of hair growth, and qualities such as kinkiness vary fromone part of the body to another, and from one person to another. All ofthese features have strong genetic determinants, as demonstrated by theheritability of these qualities.

Hair can be categorized as scalp hair, vellus hair, or androgenic(terminal) hair. Scalp hair is the hair on the head. Vellus hair is thehair on the rest of the body which has not been stimulated andtransformed by sex hormones. Androgenic hair is the hair that greatlyincreases in heaviness and rate of growth with puberty.

Most hypertrichosis is genetic, but a small number of unusual systemicdisorders can sometimes increase vellus hair. Some drugs (for instance,diazoxide, diphenylhydantoin, and minoxidil) and toxins (for instance,mercury) can induce generalized hair growth as well. Unusualhypertrichosis can also be caused by untreated infection, or bymalnutrition. For this reason, it is an occasional sign of anorexianervosa.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Lichen simplex chronicus: a skin disorder characterized by chronicitching and scratching. The constant scratching causes thick, leathery,brownish skin. The disorder may begin with something that rubs,irritates, or scratches the skin, such as clothing. This causes theperson to rub or scratch the affected area. Constant scratching causesthe skin to thicken. The thickened skin itches, causing more scratching,causing more thickening. The skin may become leathery and brownish inthe affected area. This disorder sometimes is associated with atopicdermatitis (eczema) or psoriasis. It is also associated withnervousness, anxiety, depression, and other psychological disorders.

Treatment is aimed at reducing itching and minimizing existing lesionsbecause rubbing and scratching cause the condition. The itching andinflammation may be treated with a lotion or steroid cream applied tothe affected area of the skin.

Melanoma: a malignant tumor of melanocytes and, less frequently, ofretinal pigment epithelial cells (uveal melanoma). While it representsone of the rarer forms of skin cancer, melanoma underlies the majorityof skin cancer-related deaths, and the sole effective cure is surgicalresection of the primary tumor before it achieves a thickness of greaterthan 1 mm.

Melanoma of the skin accounts for 160,000 new cases worldwide each year,and is more frequent in Caucasian men. It is particularly common inCaucasian populations living in sunny climates. About 48,000 deathsworldwide due to malignant melanoma are registered annually.

The diagnosis of melanoma requires experience, as early stages may lookidentical to harmless moles or not have any color at all. Moles that areirregular in color or shape are suspicious of a malignant melanoma or apremalignant (or pre-melanoma) lesion, which is a lesion that is likelyto progress to malignancy or melanoma over time. The treatment includessurgical removal of the tumor; adjuvant treatment; chemo- andimmunotherapy, or radiation therapy.

The most common types of melanoma include: superficial spreadingmelanoma, nodular melanoma, acral lentiginous melanoma, and lentigomaligna. Any of these types may produce melanin (and be dark in color)or not (and be amelanotic—not dark). Similarly any subtype may showdesmoplasia (dense fibrous reaction with neurotropism) which is a markerof aggressive behaviour and a tendency to local recurrence.

Melasma: (also known as chloasma or the mask of pregnancy when presentin pregnant women), melasma is a tan or dark facial skin discoloration.Although it can affect anyone, melasma is particularly common in women,especially pregnant women and those who are taking oral contraceptivesor hormone replacement therapy medications. It is also prevalent in menand women of Native American descent (on the forearms) and in men andwomen of German/Russian Jewish descent (on the face).

The symptoms of melasma include dark, irregular patches commonly foundon the upper cheek, nose, lips, upper lip, and forehead. These patchesoften develop gradually over time. Melasma does not cause any othersymptoms beyond the cosmetic discoloration. Melasma is thought to be thestimulation of melanocytes or pigment-producing cells by the female sexhormones estrogen and progesterone to produce more melanin pigments whenthe skin is exposed to sun. Women with a light brown skin type who areliving in regions with intense sun exposure are particularly susceptibleto developing this condition. Genetic predisposition is also a majorfactor in determining whether someone will develop melasma.

Palmoplantar skin: refers to the skin of the palms of the hands and thesoles of the feet, whereas non-palmoplantar skin refers to the skin onthe trunk and extremities, excluding the soles and palms. There are anumber of topographical/anatomical/site-specific differences betweenhuman skin on the sole and the palm (also called palmoplantaris) and onthe trunk (also called non-palmoplantaris) in terms of thickness andpigmentation. Not only is palmoplantar skin much thicker and less hairythan is skin in other regions of the body, but melanocytes in thoseareas are less dense and produce significantly less melanin pigment thannon-palmoplantar skin. Fibroblasts derived from palmoplantar skin inducethe expression of keratin 9, a marker for palmoplantar epidermis, innon-palmoplantar keratinocytes via mesenchymal-epithelial interactions.Further, non-palmoplantar epidermis (with no dermal components) adopts apalmoplantar phenotype when grafted on palmoplantar wounds. Finally,mRNA encoding dickkopf 1 (DKK1), an inhibitor of the canonical Wntsignaling pathway, is expressed at high levels in palmoplantarfibroblasts.

Peutz-Jeghers syndrome: an autosomal dominant disorder characterized byhyperpigmented macules on the lips and oral mucosa and gastrointestinalpolyps. The risks associated with this syndrome include increased chanceof developing cancer in multiple sites especially in thegastrointestinal tract. Other areas include the pancreas, liver, lungs,breast, ovaries, and testicles.

The average age of first diagnosis is 23, but the lesions can beidentified at birth by an astute pediatrician. Prior to puberty, themucocutaneous lesions can be found on the palms and soles. Often thefirst presentation is as a bowel obstruction from a large polyp or anintussusception, a telescoping of one loop of bowel into anothersegment.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Martin, Remington'sPharmaceutical Sciences, published by Mack Publishing Co., Easton, Pa.,19th Edition, 1995, describes compositions and formulations suitable forpharmaceutical delivery of, for instance DKK1.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for instance, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate. For topical pharmaceuticals,suitable carriers include any conventional carrier used to form atopical formulation, for instance a pharmaceutical foam, emulsion,microemulsion, cream, jelly, or liposome. Specific, non-limitingexamples of suitable carriers for topical formulations includephosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoyl phosphatidylglycerol, dioleoylphosphatidylethanolamine, phosphatidylcholine, glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether, glyceryldistearate/cholesterol/polyoxyethylene-10-stearyl ether, and propyleneglycol/ethanol/water.

Pigmented birthmark: is a blemish on the skin formed before birth,particularly one characterized by the excess deposition of melanin.Pigmented birthmarks are part of the group of pigmented skin lesionsknown as naevi. The cause of birthmarks is unknown, but may includecellular damage due to radiation or chemicals. Some types seem to run infamilies. One specific type of pigmented birthmark is a café au laitspot. The name café au lait is French for “coffee with milk” and refersto their light-brown color. While café au lait spots are usually notassociated with any medical problems, having many (three or more) suchspots is linked with neurofibromatosis and the rare McCune-Albrightsyndrome.

Polycystic ovarian syndrome: (also known clinically as Stein-Leventhalsyndrome), is an endocrine disorder that affects 5-10% of women. Itoccurs amongst all races and nationalities, is the most common hormonaldisorder among women of reproductive age, and is a leading cause ofinfertility. The principal features are lack of regular ovulation andexcessive amounts or effects of androgenic hormones. The symptoms andseverity of the syndrome vary greatly between women. While the causesare unknown, insulin resistance (often secondary to obesity) is heavilycorrelated with polycystic ovarian syndrome. Common symptoms includeoligomenorrhea, amenorrhea, infertility, generally resulting fromchronic anovulation (lack of ovulation), elevated serum levels ofandrogens, specifically testosterone, androstenedione, anddehydroepiandrosterone sulfate, causing hirsutism and occasionallymasculinization, central obesity, androgenic alopecia, acne, oily skin,seborrhea, acanthosis nigricans (dark patches of skin, tan to dark brownor black), acrochordons (skin tags), prolonged periods of PMS-likesymptoms (bloating, mood swings, pelvic pain, backaches), and sleepapnea.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The term polypeptide orprotein as used herein encompasses any amino acid sequence and includesmodified sequences such as glycoproteins. The term polypeptide isspecifically intended to cover naturally occurring proteins, as well asthose that are recombinantly or synthetically produced.

The term polypeptide fragment refers to a portion of a polypeptide thatexhibits at least one useful epitope. The phrase “functional fragmentsof a polypeptide” refers to all fragments of a polypeptide that retainan activity, or a measurable portion of an activity, of the polypeptidefrom which the fragment is derived. Fragments, for example, can vary insize from a polypeptide fragment as small as an epitope capable ofbinding an antibody molecule to a large polypeptide capable ofparticipating in the characteristic induction or programming ofphenotypic changes within a cell. An epitope is a region of apolypeptide capable of binding an immunoglobulin generated in responseto contact with an antigen. Thus, smaller peptides containing thebiological activity of DKK1, or conservative variants of DKK1, are thusincluded as being of use.

Porphyria cutanea tarda: is the most common type of porphyria. Thedisorder results from low levels of the enzyme responsible for the fifthstep in heme production. Symptoms usually begin in adulthood and resultfrom the skin becoming overly sensitive to sunlight. Areas of skinexposed to the sun develop severe blistering, scarring, changes inpigmentation, and increased hair growth. Exposed skin becomes fragileand is easily damaged. People with porphyria cutanea tarda also haveincreased iron levels in the liver. They face a higher risk ofdeveloping abnormal liver function and liver cancer. The signs andsymptoms of this condition are triggered by nongenetic factors such asalcohol abuse, excess iron, certain hormones, and viral infections.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is at risk for a disease such as vitiligo, lichen simplex chronicus,melasma, porphyria cutanea tarda, Addison's disease, acanthosisnigricans, hirsutism, Peutz-Jeghers syndrome, congenital adrenalhyperplasia, polycystic ovarian syndrome, hypertrichosis, or melanoma.An example of a subject at risk for melanoma includes a person with afamily history of skin cancer or a personal history of excessive sunexposure. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop.

Protein: a biological molecule expressed by a gene and comprised ofamino acids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell or within aproduction reaction chamber (as appropriate).

Sequence identity: the similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of a DKK1 protein, and thecorresponding cDNA sequence, will possess a relatively high degree ofsequence identity when aligned using standard methods. This homologywill be more significant when the orthologous proteins or cDNAs arederived from species that are more closely related (for example, humanand chimpanzee sequences), compared to species more distantly related(for example, human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman J. Mol. Biol. 147(1):195-197, 1981; Needleman and Wunsch J.Mol. Biol. 48: 443-453, 1970; Pearson and Lipman Proc. Natl. Acad. Sci.USA 85: 2444-2448, 1988; Higgins and Sharp Gene, 73: 237-244, 1988;Higgins and Sharp CABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res.16, 10881-10890, 1988; Huang et al. Computer Appls. in the Biosciences8, 155-165, 1992; and Pearson et al. Meth. Mol. Bio. 24, 307-331, 1994.Furthermore, Altschul et al. (J. Mol. Biol. 215:403-410, 1990) present adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215: 403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.The Search Tool can be accessed at the NCBI website, together with adescription of how to determine sequence identity using this program.

Nucleic acid sequences that do not show a high degree of identity cannevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

Skin: includes the epidermis and dermis of an animal. Mammalian skinconsists of two major, distinct layers. The outer layer of the skin iscalled the epidermis. The epidermis is comprised of the stratum corneum,the stratum granulosum, the stratum spinosum, and the stratum basale,with the stratum corneum being at the surface of the skin and thestratum basale being the deepest portion of the epidermis. The epidermisis between 50 μm and 0.2 mm thick, depending on its location on thebody.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum corneum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum corneum is filled with different lipids arranged inlattice-like formations which provide seals to further enhance theskin's permeability barrier.

Skin damage due to steroid treatment: Local side effects of topicalsteroids include skin thinning (atrophy) and stretch marks (striae),easy bruising and tearing of the skin, perioral dermatitis (rash aroundthe mouth), enlarged blood vessels (telangiectasia), susceptibility toskin infections, disguising infection (for instance, tinea incognito),and allergy to the steroid cream. The risk of these side effects dependson the strength of the steroid, the length of application, the sitetreated, and the nature of the skin problem.

Skin grafting: a type of medical grafting involving the transplantationof skin. The transplanted tissue is called a skin graft. Skin graftingis often used to treat extensive wounding or trauma, burns, ulcers, suchas diabetic foot ulcers, areas of prior infection with extensive skinloss, and specific surgeries that may require skin grafts for healing tooccur.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals. The methods disclosed hereinhave equal applications in medical and veterinary settings. Therefore,the general term “subject” is understood to include all animals,including, but not limited to, humans or veterinary subjects, such asother primates, dogs, cats, horses, and cows.

Therapeutically effective amount: A quantity of a specified compound(such as DKK1 or an equivalent thereof) required to achieve a desiredeffect in a subject being treated. For instance, this can be the amountnecessary to treat or prevent a dermatological condition in a subject,or a dose sufficient to prevent advancement, or to cause regression of adisease, or which is capable of relieving symptoms caused by a disease,such as uneven skin pigmentation, hyperpigmentation, unwanted hairgrowth, or thin skin.

Topical administration: refers to the delivery of a composition, such asa protein, to an animal by contacting, directly or otherwise, aformulation that includes the protein to all or a portion of the skin(epidermis) of an animal. The term encompasses several routes ofadministration including, but not limited to, topical and transdermaladministration. A common requirement for these modes of administrationis penetration of the skin's permeability barrier and efficient deliveryto the target tissue or stratum. In one embodiment, topicaladministration is used as a means to penetrate the epidermis and dermisand ultimately achieve systemic delivery of a protein. In anotherexample, topical administration is used as a means to selectivelydeliver a protein to the epidermis or dermis of an animal, or tospecific strata thereof.

Uneven skin pigmentation: refers to skin pigmentation that is moreintense in some areas than in other. Uneven skin pigmentation isparticularly bothersome to some individuals when it occurs on the face.

Vitiligo: also called leukoderma, is a chronic skin condition thatcauses loss of pigment, resulting in irregular pale patches of skin. Theprecise etiology of vitiligo is complex and not fully understood. Thereis some evidence suggesting it is caused by a combination ofauto-immune, genetic, and environmental factors. The populationincidence in the United States is between 1% and 2%.

Half of people with vitiligo develop patches of de-pigmented skinappearing on extremities before their 20s. The patches may grow orremain constant in size. Patches often occur symmetrically across bothsides on the body. Occasionally small areas may repigment as they arerecolonised by melanocytes. The location of vitiligo affected skinchanges over time, with some patches re-pigmenting and others becomingaffected.

In some cases, mild trauma to an area of skin seems to cause newpatches—for example around the ankles (caused by friction with shoes orsneakers). Vitiligo may also be caused by stress that affects the immunesystem, leading the body to react and start eliminating skin pigment.

Methods for removing the white patches include corticosteroids,calcineurin inhibitors, ultraviolet light, and surgery, but they are notvery effective. Other treatments include exposure to narrow band UV-Blight, which seems to blur the edges of patches, and causes freckling inthe affected areas. Immunomodulator creams such as Protopic and Elidelalso cause repigmentation in some cases.

Alternatively, some people with vitiligo opt for chemicaldepigmentation, which typically uses 20% monobenzylether ofhydroquinone. This process is irreversible and generally ends up withcomplete or mostly complete depigmentation.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present disclosure, suitable methods and materials aredescribed below. In case of conflict, the present specification,including terms, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

IV. Description of Several Specific Embodiments

A. The Effects of Dickkopf 1 (DKK1) on Gene Expression and Wnt Signalingby Melanocytes

Disclosed herein is the surprising discovery that topical application ofDKK1 induces non-palmoplantar skin (for instance, skin of the trunk) todevelop a palmoplantar phenotype. In particular, in addition to causingnon-palmoplantar skin to thicken and become less hairy, topical DKK1treatment reduced skin pigmentation. This is useful both for cosmeticreasons and for the treatment of a wide variety of conditions involvingunwanted skin pigmentation, including uneven skin pigmentation,hyperpigmentation, post-inflammatory pigmentation, fragrance dermatitis,ephelides, sun-damaged skin, vitiligo (for instance, wheredepigmentation is desired in a subject with widespread vitiligo),pigmented pigmented birthmarks, moles, lentigos, lichen simplexchronicus, melasma, porphyria cutanea tarda, Addison's disease,Peutz-Jeghers syndrome, and acanthosis nigricans.

There are a number of topographical/anatomical/site-specific differencesbetween human skin on the sole and the palm (also called palmoplantaris)and on the trunk (also called non-palmoplantaris) in terms of thicknessand pigmentation (Yamaguchi et al., J. Dermatol. Sci. 40:1-9, 2005). Notonly is palmoplantar skin much thicker than is skin in other regions ofthe body, but melanocytes in those areas are less dense and producesignificantly less melanin pigment than non-palmoplantar skin.Fibroblasts derived from palmoplantar skin induce the expression ofkeratin 9, a marker for palmoplantar epidermis, in non-palmoplantarkeratinocytes via mesenchymal-epithelial interactions (Yamaguchi et al.,J. Invest. Dermatol. 112:483-488, 1999). Further, non-palmoplantarepidermis (with no dermal components) adopts a palmoplantar phenotypewhen grafted on palmoplantar wounds (Yamaguchi et al., Arch. Dermatol.137:621-628, 2001; Yamaguchi and Yoshikawa, J. Dermatol. 28:521-534,2001). Finally, mRNA encoding dickkopf 1 (DKK1), an inhibitor of thecanonical Wnt signaling pathway, is expressed at high levels inpalmoplantar fibroblasts (Yamaguchi et al., J. Cell Biol. 165:275-285,2004).

Wnt signaling consists of a canonical pathway (which involves β-cateninand multiple protein complexes containing glycogen synthase kinase 313(GSK3β), axin, adenomatous polyposis coli and Akt) and severalnon-canonical pathways (which involve calcium, protein kinase Cα (PKCα),c-Jun N-terminal kinase and focal adhesion kinase) (Kawano and Kypta, J.Cell Sci. 116:2627-2634, 2003; Zorn, Curr Biol. 11(15):R592-5, 2001).DKK1 is a secreted antagonist of canonical Wnt signaling that interactswith Wnt receptor lipoprotein receptor-related protein 6 (LRP6; Mao etal., Nature 411:321-325, 2001; Nusse, Nature. 17; 411(6835):255-6,2001). DKK1 also interacts with the transmembrane proteins Kremen (Krn)1 and 2 and blocks canonical Wnt signaling by inducing endocytosis ofthe LRP6 complex (Mao et al., Nature; 417(6889):664-7, 2002) withoutaffecting the Wnt receptor Frizzled. DKK1 induces the formation ofectopic heads in Xenopus laevis in the presence of BMP inhibitors(Glinka et al., Nature 391:357-362, 1998) and plays critical roles inmodulating apoptosis during vertebrate limb development (especiallyinter-digit space formation) by interacting with BMP (Grotewald &Ruther, 2002).

DKK genes (including DKK1, DKK2 and DKK3) are coordinately expressed inmesodermal lineages; transcripts of DKK1 are found in defined lineages,including limb buds, heart, urogenital ridge, tailbud, palate andcraniofacial regions, whereas transcripts of DKK3 are restricted to thetrunk mesenchyme (Monaghan et al., Mech Dev. 1999 September;87(1-2):45-56, 1999).

HOX gene family members are transcription factors known to regulatepatterning in the primary and secondary axes of developing embryos. HOXgenes play critical roles in limb development (Dolle et al., EMBO J.8(5):1507-15, 1989) and their collinear regulation is similar to thatseen in the trunk: genes located in the middle of the HoxD complex areexpressed in proximal areas of the limb bud while genes located in amore 5′ direction have a more distal expression. HOX genes are alsoknown to direct topographical/anatomical/site-specific differentiationof embryonic neurons in response to levels of fibroblast growth factors(Dasen et al., Nature 425:926-933, 2003; Liu et al., Neuron.32(6):997-1012, 2001), bone morphogenetic protein (BMP: Dasen et al.,Nature 425:926-933, 2003) and retinoic acid (Schubert et al., Proc NatlAcad Sci USA 101: 10320-10325, 2004).

B. Regulation of Skin Thickness

In addition to topical DKK1's effects on skin pigmentation, topical DKK1also increases skin thickness in non-palmoplantar skin. This is usefulin treating a variety of dermatological conditions, for instance skingrafts, skin ulcers, skin abrasions or avulsion/excisions (such as thosethat leave a volume defect), injuries or predispositions to injurycaused by repetitive impacts or mechanical stress, age-related skinchanges (for instance, thinning or wrinkled skin), or skin damage due tosteroid treatment.

As described above, the epidermis in palmoplantar areas of the skin isthicker and less pigmented than in non-palmoplantar areas. Fibroblastsin palmoplantar dermis induce a thick epidermis and keratin 9 expressionin non-palmoplantar keratinocytes through mesenchymal-epithelialinteractions, whereas fibroblasts in non-palmoplantar dermis do not(Yamaguchi et al., J. Invest. Dermatol. 112:483-488, 1999).

Non-palmoplantar epidermis (excluding dermal components) can be graftedto treat palmoplantar skin defects (e.g. caused by diabetes mellitus(Yamaguchi et al., Brit. J. Dermatol. 151:1019-1028, 2004) and rheumaticdiseases (Yamaguchi et al., Brit. J. Dermatol. 152:664-672, 2005))because it can adopt a palmoplantar phenotype throughmesenchymal-epithelial interactions (Yamaguchi et al., Arch. Dermatol.137:621-628, 2001; Yamaguchi and Yoshikawa, J. Dermatol. 28:521-534,2001).

DKK1, which interacts with the Wnt receptor lipoprotein receptor-relatedprotein 6 (LRP6; Mao et al., Nature 411:321-325, 2001), is a secretedantagonist of the canonical Wnt signaling pathway, which involvesβ-catenin and multiple protein complexes containing glycogen synthasekinase 3β (GSK3β), axin, adenomatous polyposis coli (APC) and Akt(Kawano & Kypta, J. Cell Sci. 116:2627-2634, 2003).

As described above, HOX gene family members are transcription factorsregulating patterning in the primary and secondary axes of developingembryos which also control digit number and morphogenesis (Zakany etal., Science 304:1669-1672, 2004). The collinear regulation of HOX genesduring limb development is similar to that seen in the trunk: geneslocated in the middle of the HoxD complex (HoxD8) are expressed inproximal areas of the limb bud whereas genes located upstream have amore distal expression (HoxD12; Kmita et al., Nature 420:145-150, 2002).HOX genes are also known to direct topographical/site-specificdifferentiation of embryonic neurons in response to growth factors,especially those secreted by fibroblasts (Dasen et al., Nature425:926-933, 2003).

C. DKK1 Sequence Variants

There are a number of variant DKK1 sequences that can be used in lieu ofor in conjunction with known DKK1 sequences. A number of specific DKK1amino acid sequences are known. For instance, the term “DKK1” can referto any DKK1 amino acid sequence, such as GenBank™ Accession Nos.gi37183128, gi31542557, gi7110719, gi13124053, gi4545252, gi10281590,gi118092551, gi62858825, gi115313025, gi13124044, gi114630593,gi114630591, gi114630589, gi29504796, gi16306720, gi46394862, andgi5360731, all of which are specific, non-limiting examples. In additionto known DKK1 sequences, the creation of variants of these sequences isnow enabled. Other variant DKK1 sequences can be found in U.S. Pat. No.7,057,017, which is incorporated by reference in its entirety.

Variant DKK1 proteins include proteins that differ in amino acidsequence from known DKK1 sequences, but that share at least 60% aminoacid sequence identity with a known DKK1 protein. Other variants willshare at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 98% amino acid sequence identity. Manipulation of aDKK1 nucleotide sequence using standard procedures, including forinstance site-directed mutagenesis or PCR, can be used to produce suchvariants. The simplest modifications involve the substitution of one ormore amino acids for amino acids having similar biochemical properties.These conservative substitutions are likely to have minimal impact onthe activity of the resultant protein. Table 1 shows amino acids thatmay be substituted for an original amino acid in a protein, and whichare regarded as conservative substitutions.

TABLE 1 Original Residue Conservative Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu;val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

More substantial changes in enzymatic function or other protein featuresmay be obtained by selecting amino acid substitutions that are lessconservative than those listed in Table 1. Such changes include changingresidues that differ more significantly in their effect on maintainingpolypeptide backbone structure (for example, sheet or helicalconformation) near the substitution, charge or hydrophobicity of themolecule at the target site, or bulk of a specific side chain. Thefollowing substitutions are generally expected to produce the greatestchanges in protein properties: (a) a hydrophilic residue (for example,seryl or threonyl) is substituted for (or by) a hydrophobic residue (forexample, leucyl, isoleucyl, phenylalanyl, valyl or alanyl); (b) acysteine or proline is substituted for (or by) any other residue; (c) aresidue having an electropositive side chain (for example, lysyl,arginyl, or histadyl) is substituted for (or by) an electronegativeresidue (for example, glutamyl or aspartyl); or (d) a residue having abulky side chain (for example, phenylalanine) is substituted for (or by)one lacking a side chain (for example, glycine).

Variant DKK1-encoding sequences may be produced by standard DNAmutagenesis techniques, for example, M13 primer mutagenesis. Details ofthese techniques are provided in Sambrook et al. (In Molecular Cloning:A Laboratory Manual, CSHL, New York, 1989), Ch. 15. By the use of suchtechniques, variants may be created that differ in minor ways from knownDKK1 sequences. DNA molecules and nucleotide sequences that arederivatives of known DKK1 sequences, and which differ from thosedisclosed by the deletion, addition, or substitution of nucleotideswhile still encoding a protein that has at least 60% sequence identitywith known DKK1 sequences are comprehended by this disclosure. Alsocomprehended are more closely related nucleic acid molecules that shareat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98% nucleotide sequence identity with known DKK1sequences. In their most simple form, such variants may differ from thedisclosed sequences by alteration of the coding region to fit the codonusage bias of the particular organism into which the molecule is to beintroduced.

Alternatively, the coding region may be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to known DKK1 protein sequences. For example,because of the degeneracy of the genetic code, four nucleotide codontriplets—(GCT, GCG, GCC and GCA) code for alanine. The coding sequenceof any specific alanine residue within a known DKK1 protein, therefore,could be changed to any of these alternative codons without affectingthe amino acid composition or characteristics of the encoded protein.Based upon the degeneracy of the genetic code, variant DNA molecules maybe derived from the cDNA and gene sequences disclosed herein usingstandard DNA mutagenesis techniques as described above, or by synthesisof DNA sequences. Thus, this disclosure also encompasses nucleic acidsequences that encode a known DKK1 protein, but which vary from theknown nucleic acid sequences by virtue of the degeneracy of the geneticcode.

D. Functional Fragments and Variants of DKK1

Also included in the term “DKK1” are those DKK1 fragments and variantsthat maintain at least one function of the parent polypeptide, forinstance the ability to increase skin thickness, decrease skinpigmentation, or decrease hair growth. It is recognized that the gene orcDNA encoding a polypeptide can be considerably mutated withoutmaterially altering one or more of the polypeptide's functions. First,the genetic code is well known to be degenerate, and thus differentcodons encode the same amino acids. Second, even where an amino acidsubstitution is introduced, the mutation can be conservative and have nomaterial impact on the essential functions of a protein (see Stryer,Biochemistry 4th Ed., (c) W. Freeman & Co., New York, N.Y., 1995).Third, part of a polypeptide chain can be deleted without impairing oreliminating all of its functions. For example, sequence variants in aprotein, such as a 5′ or 3′ variant, may retain the full function of anentire protein. Fourth, insertions or additions can be made in thepolypeptide chain for example, adding epitope tags, without impairing oreliminating its functions (Ausubel et al., Current Protocols inMolecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998).

Other modifications that can be made without materially impairing one ormore functions of a polypeptide include, for example, in vivo or invitro chemical and biochemical modifications or the incorporation ofunusual amino acids. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquination, sumoylation, labeling, for example, with radionucleides,and various enzymatic modifications, as will be readily appreciated bythose well skilled in the art. A variety of methods for labelingpolypeptides and labels useful for such purposes are well known in theart, and include radioactive isotopes such as ³²P, ligands that bind toor are bound by labeled specific binding partners (for example,antibodies), fluorophores, chemiluminescent agents, enzymes, andantiligands. Functional DKK1 fragments and variants can be of varyinglength. For example, a fragment may consist of 10 or more, 25 or more,50 or more, 75 or more, 100 or more, or 200 or more amino acid residues.Specific, nonlimiting examples of DKK1 fragments are found in U.S. Pat.No. 7,057,017, which is incorporated by reference in its entirety.

E. Routes of Administration of DKK1

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,methods other than normal osmosis must be used. These methods are usedin some embodiments for the delivery of DKK1 through the skin'spermeability barrier to the epidermis and the dermis.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post-treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

One method for delivering biologically active substances to the skin istopical administration. Topical administration can be used as the routeof administration when local delivery of a drug is desired at, orimmediately adjacent to, the point of application of the drugcomposition or formulation. Three general types of topical routes ofadministration include administration of a drug composition to mucousmembranes, skin or eyes.

Transdermal drug delivery is a valuable route for the administration oflipid soluble therapeutics. The dermis is more permeable than theepidermis and therefore absorption is much more rapid through abraded,burned or denuded skin. Inflammation and other physiologic conditionsthat increase blood flow to the skin also enhance transdermaladsorption. Absorption via this route may be enhanced by the use of anoily vehicle (inunction) or through the use of one or more penetrationenhancers. Other effective ways to deliver drugs via the transdermalroute include hydration of the skin and the use of controlled releasetopical patches. The transdermal route provides an effective means todeliver a drug for either systemic or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field; see, for instance,Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea; see, for instance, Lee et al., p. 166),and optimization of vehicle characteristics relative to dose depositionand retention at the site of administration (see, for instance, Lee etal., p. 168) are useful methods for enhancing the transport of drugsacross mucosal sites in accordance with compositions and methods of thepresent invention.

In some embodiments, DKK1 is administered topically with one or morepenetration enhancers, for example those described below in section F.The DKK1 is generally applied in the form of a cream, lotion, ointment,tincture, liposome, emulsion, nanoformulation, microsponge formulation,or other formulation, as described below in section F, and according toan administration protocol, as outlined in section G.

F. Pharmaceutical Compositions for Topical Administration

Pharmaceutical compositions that contain DKK1 can include, but are notlimited to, solutions, emulsions, and liposome-containing formulations.These compositions are formulated for topical administration, and aregenerated from a variety of components that include, but are not limitedto, preformed liquids, self-emulsifying solids and self-emulsifyingsemisolids. DKK1-containing pharmaceutical compositions, which in someexamples are presented in unit dosage form, are prepared according toconventional techniques well known in the pharmaceutical industry. Suchtechniques include the step of bringing into association the activeingredients with one or more pharmaceutical carriers or excipients,which in some examples are liquid carriers or finely divided solidcarriers.

DKK1-containing compositions are, in some embodiments, formulated assuspensions in aqueous, non-aqueous or mixed media. In some embodiments,aqueous suspensions further contain substances which increase theviscosity of the suspension, including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. Such a suspension alsomay contain stabilizers.

In certain embodiments, a DKK1-containing pharmaceutical composition isformulated as a foam. Pharmaceutical foams include formulations such as,but not limited to, emulsions, microemulsions, creams, jellies, andliposomes. While similar in nature, these formulations vary in thecomponents and the consistency of the final product. Also useful arenanoformulations and microsponge formulations (see, for instance,Grimes, Cutis. 2004 December; 74(6):362-8.). The preparation of suchcompositions and formulations is generally known to those skilled in thepharmaceutical and formulation arts, and can be applied to theformulation of the compositions disclosed herein.

1. Emulsions

The DKK1-containing compositions disclosed herein can be prepared andformulated as emulsions. Emulsions are typically heterogeneous systemsof one liquid dispersed in another in the form of droplets, and thosedroplets usually exceed 0.1 um in diameter (see Idson in “PharmaceuticalDosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.199; Rosoff in “Pharmaceutical Dosage Forms,” Lieberman, Rieger andBanker (Eds.), 1988, volume 1, p. 245; Block in “Pharmaceutical DosageForms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 2, p. 335; andHiguchi et al., in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasicsystems comprising two immiscible liquid phases intimately mixed anddispersed with each other.

In general, emulsions may be either water in oil or oil in water. Whenan aqueous phase is finely divided into and dispersed as minute dropletsinto a bulk oily phase, the resulting composition is called a water inoil emulsion. Alternatively, when an oily phase is finely divided intoand dispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil in water emulsion. Emulsions may containadditional components in addition to the dispersed phases and the activedrug, which may be present as a solution in either the aqueous phase,the oily phase, or as a separate phase itself.

Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions. Pharmaceutical emulsionsmay also be multiple emulsions that are comprised of more than twophases such as, for example, oil in water in oil and water in oil inwater emulsions. Multiple emulsions in which individual oil droplets ofan oil in water emulsion enclose small water droplets constitute a waterin oil in water emulsion. Likewise, a system of oil droplets enclosed inglobules of water stabilized in an oily continuous provides an oil inwater in oil emulsion.

Emulsions generally are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.

Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally-occurring emulsifiers, absorption bases, and finely-dispersedsolids (see, for instance, Idson, 1988, volume 1, p. 199). Syntheticsurfactants, also known as surface active agents, have found wideapplicability in the formulation of emulsions (see, for instance,Rieger, 1988, volume 1, p. 285; Idson, 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance, and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants areclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (Rieger, 1988, volume1, p. 285).

Specific, non-limiting examples of naturally-occurring emulsifiers usedin emulsion formulations include lanolin, beeswax, phosphatides,lecithin and acacia. Specific, non-limiting examples of absorption basesthat possess hydrophilic properties such that they can soak up water toform water in oil emulsions, yet retain their semisolid consistencies,include anhydrous lanolin and hydrophilic petrolatum. Finely dividedsolids also can be used as emulsifiers, especially in combination withsurfactants and in viscous preparations. Specific, non-limiting examplesof these include polar inorganic solids, such as heavy metal hydroxides,non-swelling clays such as bentonite, attapulgite, hectorite, kaolin,montmorillonite, colloidal aluminum silicate, and colloidal magnesiumaluminum silicate, and pigments and non-polar solids such as carbon orglyceryl tristearate.

A large variety of non-emulsifying materials also can be included inemulsion formulations, and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, 1988, volume 1, p. 335; Idson, 1988, volume 1, p. 199).Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylc cellulose andcarboxypropyl cellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols, and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methylparaben, propylparaben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, boricacid and phenoxyethanol. Antioxidants are also commonly added toemulsion formulations to prevent deterioration of the formulation.Antioxidants used may be free radical scavengers such as tocopherols,alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, orreducing agents such as ascorbic acid and sodium metabisulfite, andantioxidant synergists such as citric acid, tartaric acid, and lecithin.

In some embodiments, DKK1-containing compositions are formulated asmicroemulsions. A microemulsion is a system of water, oil, andamphiphile, which is a single optically isotropic and thermodynamicallystable liquid solution (see, for instance, Rosoff, 1988, volume 1, p.245). Typically, microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Thus, microemulsionsgenerally are thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung & Shah, in: Controlled Release of Drugs:Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers,New York, pages 185-215).

Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant, andelectrolyte. Whether the microemulsion is of the water-in-oil or anoil-in-water type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,1985, p. 271). Compared to conventional emulsions, microemulsions offerthe advantage of solubilizing water-insoluble drugs in a formulation ofthermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750),decaglycerol sequioleate (S0750), and decaglycerol decaoleate (DA0750),either alone or in combination with cosurfactants. The cosurfactant,usually a short-chain alcohol such as ethanol, 1-propanol, and1-butanol, serves to increase the interfacial fluidity by penetratinginto the surfactant film and consequently creating a disordered filmbecause of the void space generated among surfactant molecules.Microemulsions may, however, be prepared without the use ofcosurfactants, and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase typically includes, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase often includes, but is not limited to, materialssuch as Captex 300, Captex 355, Capmul MCM, fatty acid esters, mediumchain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glycerylfatty acid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C8-C10 glycerides, vegetable oils, and silicone oil.

Microemulsions are of particular interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions enhance the oral bioavailability of drugs, includingpeptides (Constantinides et al., Pharmaceutical Research, 1994, 11,1385; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205).Microemulsions afford advantages of improved drug solubilization,protection of drug from enzymatic hydrolysis, enhancement of drugabsorption due to surfactant-induced alterations in membrane fluidityand permeability, ease of preparation, improved clinical potency, anddecreased toxicity (Constantinides et al., 1994, 11, 1385; Ho et al., J.Pharm. Sci., 1996, 85, 138).

Often, microemulsions form spontaneously when their components arebrought together at ambient temperature. This may be particularlyadvantageous when formulating thermolabile drugs or peptides.Microemulsions also are effective for transdermal delivery of activecomponents in both cosmetic and pharmaceutical applications.DKK1-containing microemulsion compositions and formulations willfacilitate the increased absorption of DKK1 from the skin.

The microemulsions disclosed herein also may contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of DKK1. Penetration enhancersused in microemulsions can be classified as belonging to one of fivebroad categories: surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants, as discussed above.

2. Liposomes

There are many organized surfactant structures besides microemulsionsthat are useful for the formulation of drugs. These include monolayers,micelles, bilayers and vesicles. Vesicles, such as liposomes, haveparticular advantages because of their specificity and the duration ofaction they offer. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly transformable and able topass through such fine pores. Liposomes are useful for the transfer anddelivery of active ingredients to the site of action. Because theliposomal membrane is structurally similar to biological membranes, whenliposomes are applied to a tissue, the liposomes start to merge with thecellular membranes. As the merging of the liposome and cell progresses,the liposomal contents are emptied into the cell where the active agentmay act. For topical administration, liposomes present severaladvantages over other formulations, including reduced side-effectsrelated to high systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin. In addition, liposomes can be used todeliver agents such as high-molecular weight proteins into the skin.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes that interact with negatively charged compositions toform a stable complex. The positively charged composition/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell's cytoplasm.

Liposomes that are pH-sensitive or negatively charged entrap negativelycharged compositions rather than complex with them. Since both thecomposition and the lipid are similarly charged, repulsion rather thancomplex formation occurs. Nevertheless, some of the negatively chargedcomposition is entrapped within the aqueous interior of these liposomes.

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine or dipalmitoyl phosphatidylcholine. Anionic liposomecompositions generally are formed from dimyristoyl phosphatidylglycerol,while anionic fusogenic liposomes are formed primarily from dioleoylphosphatidylethanolamine. Another type of liposomal composition isformed from phosphatidylcholine such as, for example, soybeanphosphatidylcholine, and egg phosphatidylcholine. Another type is formedfrom mixtures of phospholipid and/or phosphatidylcholine and/orcholesterol.

Non-ionic liposomal systems also are useful for the delivery of drugs tothe skin, in particular systems comprising non-ionic surfactant andcholesterol. Non-ionic liposomal formulations comprising Novasome I(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) andNovasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearylether) are particularly effective in facilitating the deposition ofvarious compositions into different layers of the skin.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates that are attractive candidates for drugdelivery vehicles. Transfersomes are lipid droplets which are so highlydeformable that they are easily able to penetrate through pores whichare smaller than the droplet. Transfersomes are adaptable to theenvironment in which they are used, for instance, they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. Surface edge-activators, usually surfactants, areadded to a standard liposomal composition to make transfersomes.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance. The nature of the hydrophilic group (alsoknown as the ‘head’) provides the most useful means for categorizing thedifferent surfactants used in formulations.

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general, their hydrophile/lipophile balance values rangefrom 2 to about 18 depending on their structure. Nonionic surfactantsinclude nonionic esters such as ethylene glycol esters, propylene glycolesters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucroseesters, and ethoxylated esters. Nonionic alkanolamides and ethers suchas fatty alcohol ethoxylates, propoxylated alcohols, andethoxylated/propoxylated block polymers are also included in this class.The polyoxyethylene surfactants are the most popular members of thenonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

Liposomal delivery systems are discussed at greater length in U.S. Pat.No. 6,841,539.

3. Penetration Enhancers

In some embodiments, various penetration enhancers are used to effectthe efficient delivery of DKK1 to the skin. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories: surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Crit. Rev. Ther.Drug Carrier Systems, 1991, p. 92). Each of these classes is describedbelow in greater detail.

Surfactants are chemical entities that, when dissolved in an aqueoussolution, reduce the surface tension of the solution or the interfacialtension between the aqueous solution and another liquid, with the resultthat absorption of proteins through the mucosa is enhanced. In additionto bile salts and fatty acids, these penetration enhancers include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, andpolyoxyethylene-20-cetyl ether, and perfluorhemical emulsions, such asFC-43.

Various fatty acids and their derivatives that act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-10 alkyl esters thereof (for instance, methyl,isopropyl and t-butyl), and mono- and di-glycerides thereof (forinstance, oleate, laurate, caprate, myristate, palmitate, stearate, andlinoleate).

Various natural bile salts and their synthetic derivatives act aspenetration enhancers, as well. The term “bile salts” includes any ofthe naturally occurring components of bile as well as any of theirsynthetic derivatives. Specific, non-limiting examples of bile saltsinclude cholic acid (or its pharmaceutically acceptable sodium salt,sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholicacid (sodium deoxycholate), glucholic acid (sodium glucholate),glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, andpolyoxyethylene-9-lauryl ether (POE).

Chelating agents are compounds that remove metallic ions from solutionby forming complexes therewith, with the result that absorption ofproteins through the mucosa is enhanced. Specific, non-limiting examplesof chelating agents include disodium ethylenediaminetetraacetate, citricacid, salicylates (for instance, sodium salicylate, 5-methoxysalicylateand homovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines).

Non-chelating non-surfactant penetration enhancing compounds arecompounds that demonstrate insignificant activity as chelating agents oras surfactants, but that nonetheless enhance absorption of proteinsthrough the skin. This class of penetration enhancers includes, forexample, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives, and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone.

Agents that enhance uptake of proteins at the cellular level also may beadded to the DKK1 compositions. For example, cationic lipids, such aslipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), are also knownto enhance the cellular uptake of proteins.

Other agents may be utilized to enhance the penetration of theadministered DKK1 protein, including glycols such as ethylene glycol andpropylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such aslimonene and menthone.

Dimethyl sulfoxide (DMSO) is another common penetrant that can be usedto promote topical absorption and uptake.

4. Excipients

A pharmaceutical carrier or excipient is a pharmaceutically acceptablesolvent, suspending agent, or any other pharmacologically inert vehiclefor delivering one or more proteins to an animal. The excipient can beliquid or solid and is selected with the planned manner ofadministration in mind so as to provide for the desired bulk,consistency, etc., when combined with a nucleic acid and the othercomponents of a given pharmaceutical composition. Typical pharmaceuticalcarriers include, but are not limited to, binding agents (for example,pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (for example, lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate); lubricants (forexample, magnesium stearate, talc, silica, colloidal silicon dioxide,stearic acid, metallic stearates, hydrogenated vegetable oils, cornstarch, polyethylene glycols, sodium benzoate, sodium acetate);disintegrants (for example, starch, sodium starch glycolate); andwetting agents (for example, sodium lauryl sulphate).

Formulations for topical administration of proteins may include sterileand non-sterile aqueous solutions, non-aqueous solutions in commonsolvents such as alcohols, or solutions of the proteins in liquid orsolid oil bases. The solutions also may contain buffers, diluents andother suitable additives. Other pharmaceutically acceptable organic orinorganic excipients suitable for non-parenteral administration that donot deleteriously react with proteins can be used. Suitablepharmaceutically acceptable excipients include, but are not limited to,water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose, polyvinylpyrrolidone and the like.

5. Other Components

DKK1 compositions additionally may contain other adjunct componentsconventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics, oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the composition, such asdyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents, and stabilizers. However, such materials, when added,should not unduly interfere with the biological activities of thecomponents of the compositions. The formulations can be sterilized and,if desired, mixed with auxiliary agents, for example lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings, and/oraromatic substances and the like that do not deleteriously interact withthe DKK1 proteins of the formulation.

In addition, aqueous suspensions may contain substances that increasethe viscosity of the suspension including, for example, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, sorbitol, and/ordextran. The suspension may also contain stabilizers.

G. Treatment Regimens

The administration of therapeutic or pharmaceutical compositions thatinclude the DKK1 proteins is described herein. In general, a subject inneed of therapy or prophylaxis is administered (topically) a compositioncomprising a DKK1 protein, commonly in a pharmaceutically acceptablecarrier, in doses ranging from 0.01 μg to 10 g per kg of body weightdepending on the age of the patient and the severity of the disorder ordisease state being treated. Dosing is dependent on severity andresponsiveness of the disease state to be treated, with the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution or prevention of the disease state isachieved. Optimal dosing schedules can be calculated from the subject'sresponse to treatment, and persons of ordinary skill can easilydetermine optimum dosages, dosing methodologies and repetition rates.

The term “treatment regimen” is meant to encompass therapeutic,palliative and prophylactic modalities of topical administration of oneor more DKK1 proteins. A particular treatment regimen may last for aperiod of time that will vary depending upon the nature of theparticular disease or disorder, its severity and the overall conditionof the patient, and may extend from multiple daily applications to twicedaily applications, daily applications, or applications every two,three, four, seven, or more days, for instance once each month or onceevery year. Following treatment, the subject is monitored for changes inhis/her condition and for alleviation of the symptoms of the disorder ordisease state. The dosage of the DKK1 composition may either beincreased in the event the subject does not respond significantly tocurrent dosage levels, or the dose may be decreased if an alleviation ofthe symptoms of the disorder or disease state is observed.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the bioactive agent is administered in maintenance doses,ranging from 0.01 μg to 10 g per kg of body weight, with a frequency offrom once or more daily to once every year.

Prophylactic treatment for high risk individuals is also disclosedherein. As used herein, the term “high risk individual” is meant torefer to an individual for whom it has been determined, for instance viaindividual or family history or genetic testing, that there is asignificantly higher than normal probability of being susceptible to theonset or recurrence of a disease or disorder. For example, a subjectcould have a personal and/or family medical history that includesfrequent occurrences of a particular disease or disorder, or could havehad such a susceptibility determined by genetic screening. As part of atreatment regimen for a high risk individual, the individual isprophylactically treated to prevent the onset or recurrence of thedisease or disorder. Prophylactically effective amounts of apharmaceutical composition typically are determined by the effect theyhave compared to the effect observed when a second pharmaceuticalcomposition lacking the active agent is administered to a similarlysituated individual.

The DKK1 compositions described herein are useful for treating and/orpreventing a variety of dermatological conditions, for instance a skingraft, an ulcer (such as a foot ulcer, particularly a diabetic footulcer), an abrasion, an injury caused by a repetitive impact ormechanical stress, age-related skin changes, skin damage due to steroidtreatment, uneven skin pigmentation, hyperpigmentation,post-inflammatory pigmentation, ephelides, fragrance dermatitis,vitiligo (for instance, where depigmentation is desired in a subjectwith widespread vitiligo), a pigmented birthmark (particularly thosecharacterized by the deposition of excess melanin), lentigos, or skinchanges due to lichen simplex chronicus, melasma, porphyria cutaneatarda, Addison's disease, Peutz-Jeghers syndrome, acanthosis nigricans,hirsutism, congenital adrenal hyperplasia, polycystic ovarian syndrome,hypertrichosis, or anorexia nervosa. Topical DKK1 administration isuseful for increasing skin thickness, decreasing skin pigmentation,decreasing unwanted hair growth, and treating or preventing melanoma.

For instance, in some embodiments the skin-thickening activities oftopical DKK1 are useful when the subject has a skin graft, an ulcer, anabrasion, an injury caused by a repetitive impact or mechanical stress,age-related skin changes, or skin damage due to steroid treatment.Following treatment, the subject is monitored for changes in skinthickness and for alleviation of the symptoms of the injury, disorder,or disease state.

In other embodiments, topical DKK1 treatment is desirable because itreduces skin pigmentation. This is useful, for instance, when thesubject has uneven skin pigmentation, hyperpigmentation,post-inflammatory pigmentation, fragrance dermatitis, sun-damaged skin,ephelides, vitiligo (for instance, where depigmentation is desired in asubject with widespread vitiligo), a pigmented birthmark, lentigos,lichen simplex chronicus, melasma, porphyria cutanea tarda, Addison'sdisease, Peutz-Jeghers syndrome, or acanthosis nigricans. Followingtreatment, the subject is monitored for changes in skin pigmentation andfor alleviation of the symptoms of the disorder, or disease state.

In still other embodiments, topical DKK1 treatment is useful because itreduces hair growth. This is desirable, for example, in subjects whowant to decrease hair growth on upper or lower extremities or onaxillary skin for cosmetic reasons. Furthermore, topical DKK1 treatmentis useful when the subject has hirsutism, congenital adrenalhyperplasia, polycystic ovarian syndrome, hypertrichosis, porphyriacutanea tarda, or anorexia nervosa.

In yet other embodiments, topical DKK1 treatment is effective as atreatment or preventive therapy for melanoma. Topical DKK1 isadministered at least daily, and can be used in conjunction with otheranti-cancer therapies, such as chemotherapy (for instance treatment withdacarbazine), immunotherapy (for instance treatment with interleukin-2,interferon, or imiquimod) as well as local perfusion, surgery, and/orradiation therapy. Treatment is generally continued until the desiredtumor regression is achieved, and maintenance treatment may be usefuleven after complete regression of the tumor.

H. Methods for Treating Melasma

Provided herein is an exemplary protocol for treating a subject withmelasma. However, one of skill in the art can see that such a treatmentprotocol is also suitable for the treatment of other hyperpigmentationconditions, for example uneven skin pigmentation, post-inflammatorypigmentation, ephelides, fragrance dermatitis, sun-damaged skin, apigmented birthmark, lentigos, lichen simplex chronicus, melasma,porphyria cutanea tarda, Addison's disease, Peutz-Jeghers syndrome,acanthosis nigricans, or when depigmentation is desired in a subjectwith widespread vitiligo.

Topical DKK1 therapy is suitable for treating acute melasma as well asfor maintaining the improvement seen following treatment of the acutecondition. Briefly, a liposome-based DKK1 formulation is appliedtopically to the affected area of the subject twice daily, generallymorning and evening. The typical dose of DKK1 ranges from 0.1 μg per kgof body weight to 1 μg per kg of body weight, depending on the severityof the condition, the age of the subject, and the sensitivity of thesubject to adverse side effects such as burning or redness. Treatment iscontinued for at least six weeks or until the desired effect isachieved. In some cases, treatment is continued for 12, 24, 36 or moreweeks. During the time of treatment, the subject is advised to avoid sunexposure.

Once the desired degree of skin lightening is achieved, DKK1 applicationis reduced to once a day. In some cases, treatment will continueindefinitely, whereas in other cases treatment is discontinued after aperiod of maintenance treatment.

I. Methods for Preventing Melanoma

This Example provides an exemplary protocol for preventing melanoma in asubject at risk for developing the condition. A subject may be at riskfor developing melanoma, for instance, if she or he has a family historyof skin cancer or a personal history of melanoma or excessive sunexposure. The disclosed method can be used to inhibit the development ofabnormal pigmented lesions (such as nevi) that sometimes are precursorsof melanoma, or can be used to treat suspicious lesions that are ofirregular shape and/or increasing size.

Briefly, a microsponge-based DKK1 formulation is applied topically tothe affected area of the subject twice daily, generally morning andevening. The typical dose of DKK1 ranges from 1 μg per kg of body weightto 10 μg per kg of body weight, depending on the age of the subject andthe sensitivity of the subject to adverse side effects such as burningor redness. In some cases, treatment begins with a lower initial dose ofDKK1 (for example 0.1 mg/kg body weight) before the full dose isadministered. Treatment is continued for at least six weeks and may becontinued for 12, 24, 36 or more weeks, or may be continuedindefinitely.

After an initial period of treatment, the DKK1 dose may be decreasedapplication is reduced to once a day. In some cases, treatment willcontinue indefinitely, whereas in other cases treatment is discontinuedafter a period of maintenance treatment.

In some embodiments, DKK1 is administered to a subject at risk ofdeveloping melanoma, particularly acral lentiginous melanoma, or to asubject who has already developed melanoma that has been surgicallyexcised, through another route of administration, for instance throughparenteral administration, targeted gene therapy, or other routes ofadministration, such as a micropump or sustained-release formulation.For instance, DKK1 can be administered in the form of a lentivirusover-expressing DKK1 whose expression is controlled by the expression ofa melanocyte-specific gene, such as tyrosinase for example. This limitsoverexpression of DKK1 to melanocytes and in melanoma cells. In theseembodiments, DKK1 is administered in a dose ranging from 0.01 μg per kgof body weight to 10 g per kg of body weight, depending on the age ofthe subject, the severity of the condition, and the sensitivity of thesubject to adverse side effects.

J. Methods for Treating Thin Skin

Provided herein is an exemplary protocol for treating a subject withthin skin, for instance due to aging or treatment with topical steroids.However, one of skill in the art can see that such a treatment protocolis also suitable for the treatment of other conditions resulting in thinskin, for example when the subject has a skin graft, an ulcer, anabrasion, or an injury caused by a repetitive impact or mechanicalstress. In some embodiments, the skin being treated is on the hands, forinstance, in age-related thinning of the skin, and in certain examples,the DKK1 is provided in the form of a hand créme or lotion.

In some embodiments, the subject has a palmoplantar burn, a foot ulcer,for instance a diabetic foot ulcer, or another type of erosion injury,such as those resulting from collagen diseases, such as systemicsclerosis, poly arthritis nodosa, and rheumatoid arthritis. These typesof skin injuries are treated, in some examples, with grafts oftrunk-derived epidermis or tissue engineered from fibroblasts, which isinduced by DKK1 to adopt a plantar phenotype. The plantar phenotyperesults in a more durable skin graft that is resistant to furtherdamage. Thus, otherwise intractable palmoplantar wounds, for instancethose with exposed bones, can be treated, for instance, with acombination of bone marrow exposure, occlusive dressing, epidermalgrafting, and treatment with DKK1. See, for instance, Atiyeh et al.,(2005) Burns, 31:944-956, for a review of methods of closing burns andother wounds; Wong et al., (2007) Br. J. Dermatol. 156:1149-1155, for areview of methods of using fibroblasts for tissue engineering; andYamaguchi et al. (2004) Br. J. Dermatol. 151:1019-1028, for methods ofhealing intractable diabetic foot ulcers with exposed bones, all ofwhich are incorporated herein by reference. In each case, the grafttissue is exposed to DKK1, either before or after transplantation, inorder to induce a durable palmoplantar phenotype.

Topical DKK1 therapy is suitable for treating acutely thinning skin orskin grafts, as well as for maintaining the improvement seen followingtreatment of the acute condition. Briefly, an emulsion-based DKK1formulation is applied topically to the affected area of the subjecttwice daily, generally morning and evening. The typical dose of DKK1ranges from 0.1 μg per kg of body weight to 1 μg per kg of body weight,depending on the severity of the condition, the age of the subject, andthe sensitivity of the subject to adverse side effects such as burningor redness. In certain examples, about 10-1,000 ng/ml DKK1 is appliedtopically, for instance, from about 50 ng/ml to about 500 ng/ml, orabout 100 ng/ml. Treatment is continued for at least six weeks or untilthe desired effect is achieved. In some cases, treatment is continuedfor 12, 24, 36 or more weeks.

Once the desired degree of skin thickening is achieved, DKK1 applicationis reduced to once a day. In some cases, treatment will continueindefinitely, whereas in other cases treatment is discontinued after aperiod of maintenance treatment.

Without further elaboration, it is believed that one skilled in the artcan, using this description, utilize the present discoveries to theirfullest extent. The following examples are illustrative only, and notlimiting of the disclosure in any way whatsoever.

Example 1 Materials and Methods Used for Evaluating the Effects of DKK1on Melanocyte Function and Proliferation

This Example provides representative materials and methods used forevaluating the effects of DKK1 on melanocyte function and proliferationthat provide the therapeutic effects described herein.

Melanocyte Cultures

Neonatal human foreskin melanocytes were obtained from CascadeBiologics, Inc. (Portland, Oreg.). Melanocyte cultures were grown inmelanocyte growth medium (MGM) consisting of Medium 154 (CascadeBiologics, Inc.) and HMGS (Cascade Biologics, Inc.). Melanocytes fromthe third to fifth passage were used in these experiments.

Microarray Procedures

Modified oligo-cDNA microarray analysis was performed as previouslydescribed with slight modifications (Yamaguchi et al., J. Cell Biol.165:275-285, 2004). Briefly, total RNA was prepared from cultured humanmelanocytes treated with or without 50 ng/ml rhDKK1 (R&D Systems Inc.,Minneapolis, Minn.) for two hours, using an RNeasy mini kit (Qiagen,Valencia, Calif.). The quality (purity and integrity) of extracted totalRNA was confirmed using an Agilent 2100 Bioanalyzer with an RNA 6000Nano Assay (Agilent Technology, Palo Alto, Calif.). Paired cDNA samples,labeled by cyanine 3- and cyanine 5-dUTP incorporation (Qiagen) duringreverse transcription (Qiagen), were hybridized simultaneously with oneoligo-DNA chip (Hs-Operon V2-vB2.2p13) as per National Cancer Institute(NCI) in-house protocol (available at http://mach1.nci.nih.gov/). Twofluorescent intensities of the oligo-DNA chip were scanned using amicroarray scanner (GenePix 4000A; Axon Instruments, Inc., MolecularDevices Corp., Sunnyvale, Calif.). Differential gene expression wasprofiled with Genepix 3.0 software and was analyzed by NCI Center forInformation Technology programs and databases. Experiments wereperformed in triplicate independently.

RT-PCR

To confirm the accuracy of oligo-cDNA microarrays, RT-PCR was performed.Oligonucleotide primers used for PCR were based on published mRNAsequences as follows: human PKCβ1 sense primer5′-agtgccaagtttgctgcttt-3′ (SEQ ID NO: 1); PKCβ1 antisense primer5′-acaatgaggacgtccctgtc-3′ (SEQ ID NO: 2); human Krn1 sense primer5′-caagtttgctgggatggagt-3′ (SEQ ID NO: 3); Krn1 antisense primer5′-tgtcaaatagggggaagctg-3′ (SEQ ID NO: 4); human LRP6 sense primer5′-gctcagagtcccagttccag-3′ (SEQ ID NO: 5); LRP6 antisense primer5′-tcccttcatacgtggacaca-3′ (SEQ ID NO: 6); human LDLR sense primer5′-aagtgcatctctcggcagtt-3′ (SEQ ID NO: 7); LDLR antisense primer5′-tgcagtttccatcagagcac-3′ (SEQ ID NO: 8); human GPR51 sense primer5′-gctgctgatcgacctgtgta-3′ (SEQ ID NO: 9); GPR51 antisense primer5′-gatggtgctgcagaagatga-3′ (SEQ ID NO: 10); human TNFRSF10A sense primer5′-agagagaagtccctgcacca-3′ (SEQ ID NO: 11); TNFSF10A antisense primer5′-ttgtgagcattgtcctcagc-3′ (SEQ ID NO: 12); human Gadd4513 sense primer5′-acagtgggggtgtacgagtc-3′ (SEQ ID NO: 13); Gadd4513 antisense primer5′-gagatgtaggggacccactg-3′ (SEQ ID NO: 14); MITF sense primer5′-agagagcgagtgcccaggcatgaac-3′ (SEQ ID NO: 15); MITF antisense primer5′-tctttggccagtgctcttgcttcag-3′ (SEQ ID NO: 16); GAPDH sense primer5′-accacagtccatgccatcac-3′ (SEQ ID NO: 17); GAPDH antisense primer5′-tccaccaccctgttgctgta-3′ (SEQ ID NO: 18). After denaturation at 94° C.for two minutes, PCR was performed for 30 cycles (30 seconds at 94° C.,one minute at 56° C. and one minute at 72° C.). All amplified productswere sequence verified (Yamaguchi et al., J. Cell Biol. 165:275-285,2004). Control reactions were performed in the absence of reversetranscriptase and were negative. Each experiment was repeated intriplicate independently.

Immunoblotting

Cultures from 100 mm dishes were solubilized in 500 μl M-PER® mammalianprotein extraction reagent (Pierce Biotechnology, Rockford, Ill.) orextraction buffer containing 1% Nonidet P 40 (Calbiochem, San Diego,Calif.), 0.01% SDS, 0.1 M Tris:HCl, pH 7.2, and Protease Inhibitorcocktail (Roche, Mannheim, Germany). Protein concentrations of extractswere measured using the BCA protein assay kit (Pierce, Rockford, Ill.).Cell extracts (1 μg) were separated on 8-16% gradient SDS polyacrylamidegels (Invitrogen Corp, Carlsbad, Calif.). After electrophoresis,proteins were transferred electrophoretically from the gels toImmobilon-P transfer membranes (Millipore, Bedford, Mass.). The filterswere incubated in the presence of antibodies to GSK3β (at 1:1,000, CellSignaling Technology, Beverly, Mass.), pGSK3β (at 1:1,000, CellSignaling Technology), PKCα (at 1:10,000, Sigma, St. Louis, Mo.),β-catenin (at 1:1,000, Cell Signaling Technology), ERK1/2 (p44/42 MAPKinase antibody) (at 1: 1,000, Cell Signaling Technology), or β-actin(at 1:3,000, AC-15, Abcam, Cambridge, Mass.) at room temperature for onehour, and were then incubated with horseradish peroxidase-linkedanti-rabbit or anti-mouse whole antibodies (at 1:1,000, AmershamBioscience, Pittsburgh, Pa.) at room temperature for one hour. Antigenswere detected using an ECL-plus Western Blotting Detection System(Amersham).

Immunocytochemical Staining

Melanocyte cultures in two-well Lab-Tek chamber slides (Nalge NuncInternational Corp., Naperville, Ill.) were processed for indirectimmunofluorescence to detect the expression of signal transductionproteins using primary antibodies to GSK3β (1:50, Cell Signaling Tech),phospho-GSK3β which is specific for GSK3β phosphorylated at Ser9 (at1:100, Cell Signaling Technology), β-catenin (at 1:50, Cell SignalingTechnology) and PKCα (at 1:1,000, Sigma).

Bound antibodies were visualized with appropriate secondary antibodies,Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (Molecular Probes, Inc.,Eugene, Oreg.) at 37° C. for 30 minutes at 1:500 dilution with 5% goatserum. DAPI (Vector Laboratories Inc., Burlingame, Calif.) was used as acounter stain. The fluorescence of green produced by Alexa 488® and ofblue by DAPI was observed and captured using a Leica DMR B/D MLDfluorescence microscope (Leica, Wetzlar, Germany) and a Dage-MTI 3CCD3-chip color video camera (Dage-MTI, Michigan City, Ind.). Fluorescencewas also observed using confocal laser scanning microscopy as describedpreviously (Hoashi et al., J. Biol. Chem. 280:14006-14016, 2005).

3-Dimensional Skin Reconstructs

The epidermal equivalent MelanoDerm® is a reconstructed skin modelprovided commercially and obtained from MatTek Corp (Ashland, Mass.,USA; www.mattek.com). Normal human keratinocytes and melanocytes used inthose reconstructs were obtained from Asian neonatal foreskin tissues.MelanoDerms were grown at the air/liquid interface of the maintenancemedium MEL-NHM-113 (MatTek Corp.), and the culture medium was renewedevery two days. Where noted, the epidermis samples were supplementedwith 100 ng/ml rhDKK1 every two days for 4 to 14 days. DKK1 wasdissolved in PBS with 0.1% BSA and the same concentrations of PBS andBSA were employed for mock-treated controls.

Immunohistochemistry

Skin specimens obtained from matched palmoplantar areas (i.e. palms;n=1, and soles; n=4) and from non-palmoplantar areas (trunk; n=5) takenfrom five adult Asian subjects (ages ranged from 31 to 47) duringcutaneous surgery. The expression of proteins related to signaltransduction was detected by indirect immunofluorescence using theprimary rabbit polyclonal antibodies described above. Mouse monoclonalantibody Ab-3 (1:100 dilution, NeoMarkers, Fremont, Calif.), which isspecific for MART1, was additionally used as a primary antibody.Secondary antibodies used were Alexa Fluor® 594 goat anti-mouse IgG(H+L) and Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (Molecular Probes,Inc.). The green fluorescence produced by Alexa 488® was superimposedover the red fluorescence produced by Alexa 594® to showco-localization.

Example 2 Expression Levels of DKK1 mRNA in Palmoplantar andNon-palmoplantar Fibroblasts

This Example demonstrates that expression levels of DKK1 mRNA areup-regulated in human palmoplantar fibroblasts compared withnon-palmoplantar fibroblasts, and that DKK1 has a number of immediateeffects on melanocytes. DKK1 expression levels were confirmed infibroblasts derived from palm or trunk skin at the protein level, usingimmunoblotting (FIG. 1A) and immunocytochemistry (FIG. 1B). Theexpression of DKK1 in three independent populations ofpalmoplantar-derived fibroblasts (Palm1, Palm2 and Palm3) wasdramatically higher than in three independent populations ofnon-palmoplantar-derived fibroblasts (Trunk1, Trunk2 and Trunk3).

Normal human melanocytes were then treated with 50 ng/ml recombinanthuman DKK1 (rhDKK1) for two hours, and total RNA was harvested andanalyzed using oligonucleotide-cDNA microarrays. The concentration ofDKK1 used was determined in previous studies (Tian et al., New Eng J Med349: 2483-2494, 2003; Yamaguchi et al., J. Cell Biol. 165:275-285,2004). The complete database of gene expression patterns are reported inSupplementary Database 1(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE5515). Inaddition, these data have been deposited as a Gene Ontology report on apublic database (http://www.ncbi.nlm.nih.gov/geo/) with a Series # of‘GSE5515’. The 30 genes with the largest increases or decreases inexpression are listed in Table 2. Highly regulated genes also includedsome receptor-related genes (Table 3), Wnt-related genes (Table 4),melanocyte marker genes (Table 5) and Homeobox (HOX)-related genes(Table 6). DKK1 had various immediate effects on melanocytes,considering that affected genes are related to such diverse processes asmitochondrial function (e.g. MRPL38 and MRPS27), melanosome trafficking(for instance, HPS4, SV2B and STX5A) and transport (for instance, MLPH),apoptosis (for instance, Gadd45β and TNFRSF10A),

Wnt signaling (for instance, β-catenin and PKC), as well as melanocytedevelopment (for instance, HOX) and differentiation (for instance,MITF).

TABLE 2 Top 30 Genes Up-Regulated in Melanocytes by DKK1 * DKK1/NS DKK1NS Gene Description 4.10 3206 782 GLIO703 Acid fibroblast growthfactor-like protein 3.71 4529 1222 ASCL3 Achaete-scute complex(Drosophila) homolog-like 3 (ASCL3) 3.58 4847 1352 GAD2 glutamatedecarboxylase 2 (pancreatic islets and brain, 65 kDa) (GAD2) 3.40 3026891 FOXI1 forkhead box I1 (FOXI1), transcript variant 2 3.39 3676 1084MBNL1 Muscleblind-like (Drosophila) 3.13 3747 1197 CAV3 caveolin 3(CAV3), transcript variant 2 3.05 8675 2840 FTL Ferritin, lightpolypeptide 2.91 5669 1946 HSPC073 HSPC073 2.79 4367 1567 CTNNB1β1-Catenin (cadherin-associated protein), 88 kDa 2.76 19609 7093 MRPL38mitochondrial ribosomal protein L38 (MRPL38), nuclear gene encodingmitochondrial protein 2.75 8914 3237 SMAD6 SMAD, mothers against DPPhomolog 6 (Drosophila) (SMAD6) 2.70 32276 11945 ANK1 Ankyrin 1,erythrocytic (ANK1), transcript variant 2 2.69 3182 1184 MAGEC1 melanomaantigen, family C, 1 (MAGEC1) 2.66 19851 7476 SV2B synaptic vesicleglycoprotein 2B (SV2B) 2.65 3305 1246 MAPT microtubule-associatedprotein tau (MAPT), transcript variant 4 2.63 15815 6018 SAFB2 Scaffoldattachment factor B2 2.61 20135 7703 UCN2 urocortin 2 (UCN2) 2.55 113004429 SMP1 small membrane protein 1 (SMP1) 2.51 4550 1811 VPS16 Proteintyrosine phosphatase, receptor type, A 2.50 7466 2988 CML66 Chronicmyelogenous leukemia tumor antigen 66 (CML66) 2.48 5439 2191 LARGELike-glycosyltransferase 2.48 34333 13862 GADD45B growth arrest andDNA-damage-inducible, beta (GADD45B) 2.47 18397 7441 MRPS27mitochondrial ribosomal protein S27 (MRPS27), nuclear gene encodingmitochondrial protein 2.46 6687 2717 PTE1 peroxisomal acyl-CoAthioesterase (PTE1), transcript variant 3 2.43 18321 7544 STX5A Syntaxin5A 2.42 7998 3299 NHP2L1 NHP2 non-histone chromosome protein 2-like 1(S. cerevisiae) (NHP2L1), transcript variant 1 2.42 5763 2382 UMP-CMPKUMP-CMP kinase (UMP-CMPK), mRNA. 2.41 27789 11535 DDX3Y DEAD(Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DDX3Y) 2.40 5956 2479SLC43A1 Solute carrier family 43, member 1 (SLC43A1) 2.39 8759 3668 TEFThyrotrophic embryonic factor 2.38 5147 2159 DTX2 Deltex homolog 2(Drosophila) (DTX2) 2.38 4719 1983 KCNJ2 potassium inwardly-rectifyingchannel, subfamily J, member 2 (KCNJ2) 0.02 130 8275 PTK6 PTK6 proteintyrosine kinase 6 0.02 235 10188 ST18 suppression of tumorigenicity 18(breast carcinoma) (zinc finger protein) (ST18) 0.03 525 19158 MBD2methyl-CpG binding domain protein 2 (MBD2), transcript variant 1 0.03183 5751 CAPS2 calcyphosphine 2 (CAPS2) 0.04 239 6389 RDS retinaldegeneration, slow (RDS) 0.05 582 10693 Similar to Kruppel-like factor 7(ubiquitous); ubiquitous Kruppel-like transcription factor 0.08 302 3958DHRS2 dehydrogenase/reductase (SDR family) member 2 (DHRS2), transcriptvariant 2 0.09 314 3674 MASP1 mannan-binding lectin serine protease 1(C4/C2 activating component of Ra-reactive factor) (MASP1), transcriptvariant 1 0.09 554 6169 ADMR adrenomedullin receptor (ADMR) 0.09 474450882 LAMC3 laminin, gamma 3 (LAMC3) 0.10 2017 19655 CA12 Carbonicanhydrase XII 0.11 452 3967 MGEA5 Meningioma expressed antigen 5(hyaluronidase) 0.12 906 7352 VCP Valosin-containing protein 0.15 11898187 SPON2 Spondin 2, extracellular matrix protein 0.15 1169 7870 VMPvesicular membrane protein p24 (VMP) 0.16 2614 16432 HIST1H2BO histone1, H2bo (HIST1H2BO) 0.17 1686 9902 MNT MAX binding protein (MNT) 0.176374 36625 BHLHB3 basic helix-loop-helix domain containing, class B, 3(BHLHB3) 0.23 4341 18614 PTPN23 protein tyrosine phosphatase,non-receptor type 23 (PTPN23) 0.24 1719 7160 RGS11 regulator ofG-protein signalling 11 (RGS11), transcript variant 1 0.25 6640 27095PTP4A1 Protein tyrosine phosphatase type IVA, member 1 0.25 2536 10173IPO8 importin 8 (IPO8) 0.27 1137 4152 SLC39A11 Solute carrier family 39(metal ion transporter), member 11 0.27 8137 29601 PFKFB46-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) 0.28 701925498 ZBTB4 zinc finger and BTB domain containing 4 (ZBTB4) 0.28 21717643 HKR1 GLI-Kruppel family member HKR1 (HKR1) 0.30 4633 15631 DHPSdeoxyhypusine synthase (DHPS), transcript variant 3 0.32 2112 6649 TORC3transducer of regulated cAMP response element-binding protein (CREB) 3(TORC3) 0.33 1897 5709 GNPDA2 glucosamine-6-phosphate deaminase 2(GNPDA2) 0.34 6899 20516 NIT2 nitrilase family, member 2 (NIT2) **Geneslisted are those whose expression levels in mock-treated melanocyteswere >3,000 (65,000 is the maximum) after 2 hr treatment with rhDKK1;data are reported as means of 3 independent experiments.

The mRNA expression levels of about 40 of those genes were validatedusing RT-PCR (FIG. 2), levels of several genes related to Wnt signalingwere confirmed to be significantly up-regulated in response to rhDKK1(for instance, β-catenin and PKC_(β1), p<0.0001), as were receptors (forinstance, Krn1(p<0.0001), LDLR (p<0.001), GPR51 (p<0.01) and TNFRSF10A(p<0.0001)) and markers of apoptosis (for instance, Gadd45(3, p=0.0016),whereas MITF was significantly down-regulated (p<0.001) by rhDKK1.Interestingly, the level of β-catenin mRNA did not correlate with theprotein level, as previously reported (Yamaguchi et al., J. Cell Biol.165:275-285, 2004).

TABLE 3 Receptor-Related Genes Up-Regulated in Melanocytes by DKK1*DKK1/NS DKK1 NS Gene Description 3.27 2385 729 GPR51 G protein-coupledreceptor 51 3.17 2172 686 PTGDR prostaglandin D2 receptor (DP) (PTGDR)3.06 1782 582 OR11A1 olfactory receptor, family 11, subfamily A, member1 (OR11A1) 2.83 2557 905 RARB retinoic acid receptor, beta (RARB),transcript variant 2, mRNA. 2.78 2249 810 TNFRSF10A tumor necrosisfactor receptor superfamily, member 10a (TNFRSF10A) 2.72 1718 631 NR3C2Nuclear receptor subfamily 3, group C, member 2 2.69 2097 780 OXTRoxytocin receptor (OXTR) 2.63 1593 605 DRD5 dopamine receptor D5 (DRD5)2.62 1028 392 KIR3DL3 killer cell immunoglobulin-like receptor, threedomains, long cytoplasmic tail, 3 (KIR3DL3) 2.53 1908 755 XCR1 chemokine(C motif) receptor 1 (XCR1) 2.51 4550 1811 VPS16 Protein tyrosinephosphatase, receptor type, A 2.45 3595 1466 PTGER3 Prostaglandin Ereceptor 3 (subtype EP3) 2.17 1810 833 THRAP2 Thyroid hormone receptorassociated protein 2 2.17 2050 944 ANTXR2 anthrax toxin receptor 2(ANTXR2) 1.99 10308 5177 EDG5 endothelial differentiation, sphingolipidG-protein-coupled receptor, 5 (EDG5) 1.97 5508 2801 GPR20 Gprotein-coupled receptor 20 (GPR20) 1.94 13038 6736 TRAF4 TNFreceptor-associated factor 4 (TRAF4), transcript variant 1 1.90 89064693 NGFR nerve growth factor receptor (TNFR superfamily, member 16)(NGFR) 1.86 2487 1337 BMPR1A bone morphogenetic protein receptor, typeIA (BMPR1A) 1.81 3949 2179 KDELR3 KDEL endoplasmic reticulum proteinretention receptor 3 (KDELR3), transcript variant 1 1.81 38066 21087TRIP6 thyroid hormone receptor interactor 6 (TRIP6) 1.70 7190 4229 LRP6low density lipoprotein receptor-related protein 6 (LRP6) 1.66 1200 725KREMEN1 kringle containing transmembrane protein 1 (KREMEN1), transcriptvariant 2 1.65 5379 3258 OR1G1 olfactory receptor, family 1, subfamilyG, member 1 (OR1G1) 1.63 23658 14556 PTPN11 protein tyrosinephosphatase, non-receptor type 11 (Noonan syndrome 1) (PTPN11) 1.6245217 27857 SCARB2 scavenger receptor class B, member 2 (SCARB2) 1.625749 3558 LDLR low density lipoprotein receptor (familialhypercholesterolemia) (LDLR) 1.59 15817 9966 RARA retinoic acidreceptor, alpha (RARA) 1.56 36705 23574 KDELR2 KDEL (Lys-Asp-Glu-Leu)endoplasmic reticulum protein retention receptor 2 (KDELR2) (1.35) 1471610905 MC1R melanocortin 1 receptor (alpha melanocyte stimulating hormonereceptor) (MC1R) (0.94) 1193 1263 KREMEN2 kringle containingtransmembrane protein 2 (KREMEN2), transcript variant 2 *Genes listedare those whose expression levels in DKK1-treated melanocyteswere >1,000 (65,000 is the maximum) after 2 hr treatment with rhDKK1;data for genes with DKK1/NS ratio >1.45 are reported as means of 3independent experiments.

TABLE 4 Wnt Related Genes Up-Regulated in Melanocytes by DKK1* DKK1/NSDKK1 NS Gene Description 3.37 2502 742 CXXC4 CXXC finger 4 (CXXC4) 3.121405 451 WNT16 wingless-type MMTV integration site family, member 16(WNT16), transcript variant 2 2.79 4367 1567 CTNNB1 β1-Catenin(cadherin-associated protein), 88 kDa 2.52 1127 447 WNT9B wingless-typeMMTV integration site family, member 9B (WNT9B) 2.51 1545 615 PRKCBP1Protein kinase C binding protein 1 2.32 6115 2633 PPP2R2C proteinphosphatase 2 (formerly 2A), regulatory subunit B (PR 52), γ isoform(PPP2R2C), transcript variant 2 2.21 1582 717 BCL9 B-cell CLL/lymphoma 9(BCL9) 2.19 1529 697 WNT3A wingless-type MMTV integration site family,member 3A (WNT3A) 2.08 2335 1123 SOX17 SRY (sex determining regionY)-box 17 (SOX17) 2.07 1502 725 DVL2 dishevelled, dsh homolog 2(Drosophila) (DVL2) 2.06 25580 12391 YY1 YY1 transcription factor (YY1)1.99 1658 833 WNT3 wingless-type MMTV integration site family, member 3(WNT3) 1.93 1565 810 PRKX protein kinase, X-linked (PRKX) 1.89 2169111480 PRICKLE1 prickle-like 1 (Drosophila) (PRICKLE1) 1.87 1665 890WNT10B wingless-type MMTV integration site family, member 10B (WNT10B)1.87 8377 4478 DKK1 dickkopf homolog 1 (Xenopus laevis) (DKK1) 1.85 1458786 PRKCB1 Protein kinase C, β1 1.84 2255 1223 WIF1 WNT inhibitoryfactor 1 (WIF1) 1.77 8056 4543 CSNK1G3 casein kinase 1, gamma 3(CSNK1G3) 1.77 1173 663 CCND2 cyclin D2 (CCND2) 0.31 424 1384 SIAH1Seven in absentia homolog 1 (Drosophila) 0.37 760 2076 WNT5AWingless-type MMTV integration site family, member 5A 0.49 13467 27466WNT7A wingless-type MMTV integration site family, member 7A (WNT7A) 0.50577 1165 WISP1 WNT1 inducible signaling pathway protein 1 0.51 771115017 CSNK1G2 casein kinase 1, gamma 2 (CSNK1G2) 0.52 1105 2140 PRKCIprotein kinase C, iota (PRKCI) 0.53 3251 6080 PRKCABP protein kinase C,alpha binding protein (PRKCABP) 0.58 3343 5731 ROCK1 Rho-associated,coiled-coil containing protein kinase 1 (ROCK1) 0.59 681 1147 PLCG1phospholipase C, gamma 1 (PLCG1), transcript variant 2 0.60 3521 5906SMAD4 SMAD, mothers against DPP homolog 4 (Drosophila) (SMAD4) 0.6121107 34437 MITF microphthalmia-associated transcription factor (MITF),transcript variant 5 0.61 12375 20423 CSNK1A1 casein kinase 1, alpha 1(CSNK1A1) 0.61 4983 8217 CUL1 cullin 1 (CUL1) 0.61 1490 2449 PPP3CCProtein phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform(calcineurin A gamma) 0.61 1286 2100 TCF7L2 transcription factor 7-like2 (T-cell specific, HMG-box) (TCF7L2) 0.63 7224 11484 PKN2 proteinkinase N2 (PKN2) 0.65 5674 8763 ROCK2 Rho-associated, coiled-coilcontaining protein kinase 2 (ROCK2) 0.65 1499 2300 VANGL2 PREDICTED:Homo sapiens vang-like 2 (van gogh, Drosophila) (VANGL2) 0.66 4450 6699MAPK9 Mitogen-activated protein kinase 9 (MAPK9), transcript variant 2*Genes listed are those whose expression levels in mock-treatedmelanocytes were >1,000 (65,000 is the maximum) after 2 hr treatmentwith rhDKK1; data for genes with DKK1/NS ratio <0.70 are reported asmeans of 3 independent experiments.

TABLE 5 Melanogenic Markers Up-Regulated in Melanocytes by DKK1* DKK1/NSDKK1 NS Gene Description 2.74 2947 1076 LOC51152 Melanoma antigen 2.693182 1184 MAGEC1 melanoma antigen, family C, 1 (MAGEC1) 2.06 8469 4105MAGEC2 melanoma antigen, family C, 2 (MAGEC2) 1.99 1705 855 BAGE4 Bmelanoma antigen family, member 4 (BAGE4) 1.96 21576 11021 MAGEF1melanoma antigen, family F, 1 (MAGEF1) 1.71 2557 1495 MLPH Melanophilin1.69 1013 598 MAGEC3 melanoma antigen, family C, 3 (MAGEC3), transcriptvariant 2 1.66 50952 30701 MATP membrane associated transporter (MATP)1.58 1050 664 AIM1 PREDICTED: Homo sapiens absent in melanoma 1 (AIM1)1.56 5957 3816 HPS4 Hermansky-Pudlak syndrome 4 (HPS4), transcriptvariant 2 0.48 635 1311 MIA2 melanoma inhibitory activity 2 (MIA2) 0.6121107 34437 MITF microphthalmia-associated transcription factor (MITF),transcript variant 5 0.62 1285 2062 RPS6KA1 ribosomal protein S6 kinase,90 kDa, polypeptide 1 (RPS6KA1) 0.64 15172 23576 DDT D-dopachrometautomerase (DDT) 0.70 2426 3489 MCAM melanoma cell adhesion molecule(MCAM) 0.70 1043 1499 BCL2 B-cell CLL/lymphoma 2 *Genes listed are thosewhose expression levels in mock-treated melanocytes were >1,000 (65,000is the maximum) after 2 hr treatment with rhDKK1; data for genes withDKK1/NS ratio <0.70 are reported as means of 3 independent experiments.

Example 3

DKK1 and Expression Patterns of Proteins Related to Wnt SignalingPathways

This example illustrates the effects of DKK1 on the expression patternsof proteins related to Wnt signaling pathways in more detail. Cellextracts were harvested after two hours (FIG. 3A) and after 5 days (FIG.3B) of treatment with rhDKK1 and analyzed them by Western blotting.Overall, GSK3β expression was unchanged at two hours but was slightlydown-regulated after five days of treatment with rhDKK1. However, GSK3βphosphorylated at Ser9 was dramatically up-regulated in response torhDKK1 within two hours, and that was also seen after five days oftreatment. PKCα expression was also increased at both time pointsexamined. As previously reported, β-catenin expression wasdown-regulated after two hours or five days of rhDKK1 treatment.Consistent results were confirmed by immunocytochemistry of rhDKK1- ormock-treated melanocytes (FIG. 4).

TABLE 6 HOX Related Genes Up-Regulated in Melanocytes by DKK1* DKK1/NSDKK1 NS Gene Description 2.71 2335 860 HOXA4 homeo box A4 (HOXA4) 2.231398 628 HOXB9 Homeo box B9 2.17 2405 1111 HOXA10 homeo box A10(HOXA10), transcript variant 2 1.93 1148 594 PHTF1 Putative homeodomaintranscription factor 1 1.90 1607 847 PROP1 prophet of Pit1, paired-likehomeodomain transcription factor (PROP1) 1.78 11115 6261 MEOX1Mesenchyme homeo box 1 1.76 7694 4364 PHTF1 putative homeodomaintranscription factor 1 (PHTF1) 1.76 1483 842 HOXB9 homeo box B9 (HOXB9)1.70 7989 4708 PKNOX2 PBX/knotted 1 homeobox 2 (PKNOX2) 1.62 1541 951DLX4 distal-less homeobox 4 (DLX4), transcript variant 2 1.59 2565 1612PHOX2A Paired-like (aristaless) homeobox 2a 1.58 1033 652 LHX3 LIMhomeobox 3 (LHX3), transcript variant 1 1.56 5171 3307 HOXB13 homeo boxB13 (HOXB13) 1.53 1802 1181 VAX2 ventral anterior homeobox 2 (VAX2) 1.524812 3158 PHC1 polyhomeotic-like 1 (Drosophila) (PHC1) 1.46 1048 716ZHX3 zinc fingers and homeoboxes 3 (ZHX3) 1.46 1196 817 HOPHomeodomain-only protein 0.26 567 2186 LOC360030 Homeobox C14 0.41 6071487 ISL1 ISL1 transcription factor, LIM/homeodomain, (islet-1) (ISL1)0.44 447 1025 DUX2 Double homeobox, 2 0.48 2649 5519 ZHX2 zinc fingersand homeoboxes 2 (ZHX2) 0.48 4075 8468 HOXB7 homeo box B7 (HOXB7) 0.55700 1261 HESX1 homeo box (expressed in ES cells) 1 (HESX1) 0.57 674 1185IPF1 insulin promoter factor 1, homeodomain transcription factor (IPF1)0.58 1373 2364 HOP homeodomain-only protein (HOP), transcript variant 20.62 1518 2461 ZHX1 Zinc fingers and homeoboxes 1 0.62 996 1597 HMX1homeo box (H6 family) 1 (HMX1) 0.64 1002 1565 PHOX2B paired-likehomeobox 2b (PHOX2B) 0.66 3887 5918 CHERP calcium homeostasisendoplasmic reticulum protein (CHERP) 0.66 3244 4920 ASH2L ash2 (absent,small, or homeotic)-like (Drosophila) (ASH2L) 0.67 4117 6182 PHTF2putative homeodomain transcription factor 2 (PHTF2) 0.67 933 1391 HOXB2homeo box B2 (HOXB2) 0.69 844 1230 HOXA5 homeo box A5 (HOXA5) 0.69 10581524 HOXD8 homeo box D8 (HOXD8) 0.70 804 1148 DMBX1diencephalon/mesencephalon homeobox 1 (DMBX1), transcript variant 1*Genes listed are those whose expression levels in mock-treatedmelanocytes were >1,000 (65,000 is the maximum) after 2 hr treatmentwith rhDKK1; data for genes with DKK1/NS ratio <0.70 are reported asmeans of 3 independent experiments.

Example 4

Secretion of DKK1 by Fibroblasts is Responsible for the PhysiologicalDifferences Between Palmoplantar Skin and Non-Palmoplantar Skin

This Example demonstrates that secretion of DKK1 by fibroblasts isresponsible for the physiological differences observed betweenpalmoplantar skin and non-palmoplantar skin. Three-dimensional skinreconstructs (termed MelanoDerm) were used that consist of normal humankeratinocytes and melanocytes grown at the air/liquid interface of themaintenance medium in the presence or absence of 100 ng/ml rhDKK1 forfour to 14 days (FIG. 5). The immunostaining of these skin reconstructswas consistent with the findings of melanocyte cultures, as shown above,and revealed decreased expression of β-catenin and GSK3β but increasedexpression of PKCα and Ser9-phosphorylated GS K3β.

These in vitro results were confirmed using immunohistochemistry ofhuman skin in situ. Melanocytes in palmoplantar skin, which are adjacentto dermal fibroblasts with high expression levels of DKK1, showedrelatively low expression of GSK3β (FIG. 6), but high expression ofGSK3β phosphorylated at Ser 9 and of PKCα. In contrast, levels ofβ-catenin expression were decreased in melanocytes in palmoplantarepidermis.

Example 5

Effects of DKK1 on Melanocyte Function and Proliferation, a Summary

This Example summarizes and expands on the data presented in theprevious three Examples, and clarifies the mechanisims by which DKK1 canlighten pigmented skin. As described above, Western blotting andimmunohistochemistry were used to confirm that DKK1 expression issignificantly higher in human fibroblasts derived from palmoplantar skin(which is unpigmented) compared with those derived from non-palmoplantarskin. The effects of rhDKK1 on human melanocytes were then demonstratedusing a multidisciplinary approach that included gene profile analysisand the investigation of signal transduction protein expressionpatterns. The expression of a large number of Wnt-related genes(including PKCβ1, Krn1 and LRP6) is quickly up-regulated in melanocytesin response to rhDKK1. Genes encoding receptors other than the knownDKK1 receptors (Krn1 and LRP6) also show up-regulated expression withintwo hours after treatment with rhDKK1, suggesting that low-densitylipoprotein receptor (LDLR), GPR51 and TNFRSF10A (also known as TRAILR1) are responsive to DKK1.

LDLR is related to LRP6, which has a DKK1 binding site (Mao et al.,Nature 411:321-325, 2001), with an alignment score of 706 by ClustalW atthe amino acid level and with a score of 230 bits by BlastP. GPR51 (alsoknown as GABABR2 (Li et al., J Invest Dermatol 123:622-633, 2003)) isrelated to MC¹R, a critical receptor expressed specifically bymelanocytes (Rouzaud et al., Mutat Res 571: 133-152, 2005), since bothof them are G-coupled protein receptors with an alignment score of 6365by ClustalW at the basic pair level. TNFRSF10A is known as a deathreceptor that leads to p53-independent apoptosis via caspase cleavage(Xu and El-Deiry, Biochem Biophys Res Commun 269: 179-190, 2000, 2000),and its up-regulation indicates that DKK1 induces melanocyte apoptosisvia this receptor. The concept that DKK1 induces apoptosis inmelanocytes also derives from the finding that the expression ofGadd4513, which induces apoptosis via the p38 MAPK pathway (Sarkar etal., Proc Natl Acad Sci USA 99: 10054-10059, 2002), is also up-regulatedin melanocytes treated with rhDKK1. In other words, the inhibition ofmelanocyte growth by DKK1 likely involves the up-regulated expression ofTNFRSF10A and/or Gadd45β.

The microarray analyses also indicate numerous other candidate geneswhich help explain the suppression of melanocyte growth and/or functionby DKK1. As examples, vitiligo is an acquired pigmentary disordercharacterized by progressive areas of depigmenting skin which resultfrom the loss of melanocytes in those hypopigmented regions. Vitiligohas been associated with thyroid disorders including Hashimotothyroiditis and Graves disease (Grimes, J Amer Med Assoc 293: 730-735,2005, 2005), and has also been associated with oxidative stress, inparticular with increased levels of intracellular reactive oxygenspecies regulated by mitochondria (Dell'Anna et al., J Invest Dermatol117: 908-913, 2001). Expression levels of thyrotrophic embryonic factorand mitochondrial ribosomal proteins are increased>2.3-fold by treatmentwith rhDKK1 which helps explain increased melanocyte toxicity inDKK1-rich tissue. The melanocyte toxicity would be expected to decreasepigmentation.

Trafficking of melanosomal proteins within melanocytes plays criticalroles in regulating the synthesis, deposition and distribution ofmelanin in melanosomes (Hoashi et al., J. Biol. Chem. 280:14006-14016,2005; Valencia et al., J Cell Sci in press, 2006). rhDKK1 up-regulatesthe expression of caveolin 3, SVG2B, melanophilin and syntaxin 5A, whichindicates that DKK1 regulates the subcellular trafficking of proteinseither directly or indirectly. rhDKK1 also up-regulates the expressionof MATP, a protein critical to the correct processing of tyrosinase(Costin et al., J Cell Sci 116: 3203-3212, 2003), thus thedifferentiation of melanocytes may be influenced by DKK1 at many levels.

rhDKK1 also affects the expression of numerous genes related with Wntand HOX functions in melanocytes (see Tables 4 and 5, respectively).Thus, DKK1 could be an important regulator of those pathways consideringtheir importance to melanocyte function (Bachmann et al., Clin CancerRes 11: 8606-8614, 2005; Dunn et al., Pigment Cell Res 18: 167-180,2005; Knight et al., Devel Dynam 229: 87-98, 2004; Takeda et al., J BiolChem 275: 14013-14016, 2000). Further, rhDKK1 enhances the expression ofacid fibroblast growth factor-like protein, which may function similarto fibroblast growth factor in terms of regulating HOX genes (Dasen etal., Nature 425:926-933, 2003; Liu et al., Neuron. 32(6):997-1012, 2001)and melanocyte growth (Berking et al., Amer J Path 158: 943-953, 2001;Dotto et al., J Cell Biol 109: 3115-3128, 1989; Halaban et al., J CellBiol 107: 1611-1619, 1988; Hirobe, Development 114: 435-445, 1992).

To further explain the manner in which DKK1 decreases melanocytefunction, key proteins in Wnt signaling pathways were investigated. Thefocus was on those genes since DKK1 is an inhibitor of the canonical Wntsignaling pathway (Kawano and Kypta, J. Cell Sci. 116:2627-2634, 2003),which is actively involved in regulating MITF function (Shibahara etal., Pigment Cell Res 13: 98-102, 2000; Yasumoto et al., EMBO J. 21:2703-2714, 2002). MITF is considered the master regulator of melanocytefunction, and regulates not only melanocyte proliferation but also theirproduction of melanin (Kim et al., J Cell Sci 116: 1699-1706, 2003;McGill et al., Mech Devel 87: 45-56, 2002). Hence the effect of DKK1 onWnt signaling reveals its widespread effect on the development ofpigmentation.

rhDKK1 suppresses the expression of β-catenin and MITF (Yamaguchi etal., J. Cell Biol. 165:275-285, 2004), and the overall expression ofGSK3β decreases in response to rhDKK1, whereas that of GSK3βphosphorylated at Ser9 increases. GSK3β is a unique protein in that itis inactivated by phosphorylation (Cohen and Frame, Nature Rev: Mol.Cell. Biol. 2:769-776, 2001). The finding that rhDKK1 inhibits β-cateninand GSK3β expression helps to explain the inhibitory effects of DKK1 onMITF expression since β-catenin and GSK3β enhance MITF activity at thepromoter level (through the activation of LEF/TCF (Arias et al., CurrOpin Genet Devel 9: 447-454, 1999)) and at the post-transcriptionallevel (by the phosphorylation at Ser298 (Takeda et al., Hum Mol Gen 9:125-132, 2000)). However, multiple protein complexes, which contain notonly Axin, adenomatous polyposis coli and Akt, but also GSK3β(http://www.stanford.edu/̂rnusse), suppress the expression of β-cateninby inhibiting its accumulation through the canonical Wnt signalingpathway (Cohen and Frame, Nature Rev: Mol. Cell. Biol. 2:769-776, 2001;Kawano and Kypta, J. Cell Sci. 116:2627-2634, 2003; Zorn, Curr Biol.11(15):R592-5, 2001). Since GSK3β is inactivated via several signalingpathways (Cohen and Frame, Nature Rev: Mol. Cell. Biol. 2:769-776, 2001;Ding et al., J Biol Chem 275: 32475-32481, 2000) and since Wnt-5ainhibits the canonical Wnt pathway by promoting the GSK3β-independentdegradation of β-catenin (Topol et al., J. Cell Biol. 162:899-908,2003), the canonical Wnt signaling pathway does not require thephosphorylation of GSK3β at Ser9, but the non-canonical Wnt signalingpathway does involve that phosphorylated form of GSK3β.

Interestingly, there was an increase in β-catenin mRNA expression afteronly two hours of rhDKK1 treatment, but a decrease in β-catenin protein,which was followed by the decreased expression of β-catenin mRNA afterfive days of rhDKK1 treatment. The rapid decrease in expression ofβ-catenin protein after two hours of rhDKK1 treatment (Yamaguchi et al.,J. Cell Biol. 165:275-285, 2004) might have transiently enhanced themRNA expression level of β-catenin. Additionally, rhDKK1 enhances theexpression of PKCα, which inactivates GSK3β (Chen et al., J Biol Chem275: 17894-17899, 2000; Fang et al., Mol. Cell. Biol. 22:2099-2100,2002) and induces apoptosis via p38 MAPK (Tanaka et al., J Biol Chem278: 33753-33762, 2003; similar to Gadd45 as described above; Sarkar etal., Proc Natl Acad Sci USA 99: 10054-10059, 2002), which indicates thatDKK1 may take its own pathway and/or the non-canonical Wnt signalingpathway to inhibit GSK3β. Taken together, the decreased activities ofβ-catenin and GSK3β via phosphorylation at Ser9 and the up-regulatedexpression of PKCα may account for the decreased expression levels ofMITF in melanocytes responding to DKK1, and their decreased growth anddifferentiation, and a decrease in pigmentation.

In conclusion, DKK1, a secretory protein expressed at relatively highlevels by fibroblasts in palmoplantar skin, has various depigmentingeffects on melanocytes. Microarray analyses revealed a large number ofup-regulated or down-regulated genes in melanocytes which respondquickly to treatment with rhDKK1. DKK1-responsive genes include thoseencoding receptors (such as LDLR, GPR51 and TNFSF10A), for apoptosis(such as Gadd45β), for melanosome trafficking and transport (such asHPS4, SV2B, STX5A and MLPH), and for Wnt and HOX signaling. Thedecreased expression levels of MITF in melanocytes treated with rhDKK1may result from the decreased activity of β-catenin and of GSK3β viaphosphorylation at Ser9 and from the up-regulated expression of PKCα.The sum of those activities results in the decreased density ofmelanocytes in palmoplantar skin and the hypopigmentation of thattissue.

Example 6 Methods Used to Demonstrate Regulation of Skin Thickness

This Example illustrates the materials and methods used to demonstrateDKK1 regulation of skin thickness. DKK1 inhibits melanocyte growth andfunction via the suppression of β-catenin and microphthalmia-associatedtranscription factor (MITF; Yamaguchi et al., J. Cell Biol. 165:275-285,2004). Since keratinocytes also play a major role in skin structure andpigmentation, the effects of DKK1 were also significant. The effects ofDKK1 on melanin uptake by keratinocytes and on keratinocyte geneexpression profiles and Wnt signaling pathways were measured todetermine whether high levels of expression and secretion of DKK1 bypalmoplantar fibroblasts accounts for the thick and hypopigmentedphenotype of skin on the soles and the palms.

Keratinocyte and HaCaT Cell Cultures

Neonatal human foreskin keratinocytes were obtained from CascadeBiologics, Inc., Portland, Oreg. Keratinocyte cultures were grown inkeratinocyte growth medium consisting of Medium 154 and HKGS (both fromCascade Biologics, Inc.). Keratinocytes from the third to fifth passagewere used in these experiments. Immortalized human HaCaT keratinocytes(Boukamp et al., J Cell Biol 106:761-771, 1988) were used for melaninuptake experiments, and were cultured in 10% FBS/Dulbecco's modifiedEagle medium (DME).

Reconstructed Skin

The epidermal equivalent MelanoDerm® was obtained from MatTek Corp(Ashland, Mass., USA). Normal human keratinocytes and melanocytes wereobtained from Asian neonatal foreskin tissues. MelanoDerms were grown atthe air/liquid interface of the maintenance medium MEL-NHM-113 (MatTekCorp.), and the culture medium was renewed every two days. Where noted,the epidermis samples were supplemented with 100 ng/ml rhDKK1 (R&DSystems, Minneapolis, Minn., USA) every two days for 4 to 10 days.rhDDK1 was dissolved in PBS with 0.1% BSA. The same concentrations ofPBS and BSA were employed for mock-treated controls.

Histochemistry

Skin specimens were obtained from palmoplantar areas (palms and soles)and from non-palmoplantar areas (trunk) and were taken from 5 adultAsian subjects (ages ranged from 31 to 47) during cutaneous surgery.Skin and epidermal equivalent samples were embedded in paraffin andsections were cut using standard techniques. The sections weredeparaffinized in xylene and hydrated through a series of gradedethanols. Specimens were observed following haematoxylin-eosin staining,Fontana-Masson staining and immunohistochemistry. The thickness ofepidermis and more specifically the thickness of the stratum corneum wasmeasured with Scion Image Software (Scion Corp, Frederick, Md., USA).Melanin content was measured after Fontana-Masson staining and was alsoanalyzed by Scion Image Software (Scion Corp). The expression of proteinwas detected by indirect immunofluorescence using primary antibodies asfollows. Mouse monoclonal antibody against keratin 9 (1:20, Abcam,Cambridge, Mass., USA), rabbit polyclonal against β-catenin (1:50, CellSignaling, Danvers, Mass., USA) and rabbit polyclonal against PAR2(1:1,000; Scott et al., J. Invest. Dermatol. 117:1412-14202001).Secondary antibodies used were Alexa Fluor 594 goat anti-mouse IgG(H+L), Alexa Fluor 488 goat anti-mouse IgG (H+L) and Alexa Fluor 488goat anti-rabbit IgG (H+L). DAPI (Vector, Burlingame, Calif., USA) wasused as a counter-stain. Fluorescence was observed and captured using aLeica DMR B/D MLD fluorescence microscope (Leica, Wetzlar, Germany) anda Dage-MTI 3CCD 3-chip color video camera (Dage-MTI, Michigan City,Ind.).

Transfection and Melanin Uptake

Transfection studies were performed using a DKK1 expressing plasmid,pcDNA3.1(−)-DKK1 (Yamaguchi et al., J. Cell Biol. 165:275-285, 2004),and the pcDNA3.1 vector alone as the control. Transfection was performedusing lipofection for keratinocytes using lipofectamine 2000(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. Keratinocytes were seeded at 60% confluence 16 hours priorto transfection in KGM or in 10% FBS/DME. The amount of DNA used foreach transfection was 2 μg per 1×10⁶ cells. After 5 days, transfectedcells were harvested for various analyses including melanin uptakeassay, immunocytochemistry and western blotting. The transfectionefficiency was 70% as determined by the pEGFP-C1 vector (BD BiosciencesClontech, Palo Alto, Calif.) and/or a β-Gal staining kit (Invitrogen).Melanin granules (2 μg/ml) purified from MNT1 melanoma cells (Kushimotoet al., Proc. Natl. Acad. Sci. USA 98:10698-10703, 2001) was added 1dbefore the harvest.

Microarray Procedures

Modified oligo-DNA microarray analysis was performed as previouslydescribed (Yamaguchi et al., J. Cell Biol. 165:275-285, 2004). Briefly,total RNA was prepared from cultured human keratinocytes treated with orwithout 50 ng/ml rhDKK1 (R&D Systems, Minneapolis, Minn.) for 2 hours,using an RNeasy mini kit (Qiagen, Valencia, Calif.). The quality (purityand integrity) of extracted total RNA was confirmed using an Agilent2100 Bioanalyzer with an RNA 6000 Nano Assay (Agilent Technology, PaloAlto, Calif.). Paired cDNA samples, labeled by cyanine 3- and cyanine5-dUTP incorporation (Qiagen) during reverse transcription (Qiagen),were hybridized simultaneously with one oligo-DNA chip (Hs-OperonV2-vB2.2p13) as per NCI in-house protocol (available athttp://mach1.nci.nihgov1). Two fluorescent intensities of the oligo-DNAchip were scanned using a microarray scanner (GenePix 4000A; AxonInstruments, Inc., Sunnyvale, Calif.). Differential gene expression wasprofiled with Genepix 3.0 software and was analyzed by NCI Center forInformation Technology (CIT) programs and databases. All experimentswere performed in triplicate independently.

RT-PCR

To confirm the validity of oligo-DNA microarray results, RT-PCR wasperformed. The oligonucleotide primers for PCR were based on publishedmRNA sequences and were as follows: human KLEIP sense primer5′-tggctgtgttaggagggttc-3′ (SEQ ID NO: 19); KLEIP antisense primer5′-ctactgccatgagctgtcca-3′ (SEQ ID NO: 20); human GJB6 sense primer5′-ggcgaggagagaagaggaat-3′ (SEQ ID NO: 21); GJB6 antisense primer5′-caactctgccacgttaagca-3′ (SEQ ID NO: 22); human Snrpn sense primer5′-gttttgggtctggtgttgct-3′ (SEQ ID NO: 23); Snrpn antisense primer5′-gacctctaatgcctggtgga-3′ (SEQ ID NO: 24); human BMP21K sense primer5′-cacgccaactagcacaaaga-3′ (SEQ ID NO: 25); BMP21K antisense primer5′-aattcgactggttgggactg-3′ (SEQ ID NO: 26); MITF sense primer5′-agagagcgagtgcccaggcatgaac-3′ (SEQ ID NO: 15); MITF antisense primer5′-tctttggccagtgctcttgcttcag-3′ (SEQ ID NO: 16); human P4HA2 senseprimer 5′-tgtcaaactgacaccccgta-3′ (SEQ ID NO: 27); P4HA2 antisenseprimer 5′-atttactcgggccacaacag-3′ (SEQ ID NO: 28); human Tulp3 senseprimer 5′-acaccgtggatactgcttcc-3′ (SEQ ID NO: 29); Tulp3 antisenseprimer 5′-ccgatccattccccttttat-3′ (SEQ ID NO: 30); human PAR2 senseprimer 5′-tgctagcagcctctctctcc-3′ (SEQ ID NO: 31); PAR2 antisense primer5′-cttcaaggggaaccagatga-3′ (SEQ ID NO: 32); GAPDH sense primer5′-accacagtccatgccatcac-3′ (SEQ ID NO: 17); GAPDH antisense primer5′-tccaccaccctgttgctgta-3′ (SEQ ID NO: 18). After denaturation at 94° C.for two minutes, PCR was performed for 30 cycles (30 seconds at 94° C.,one minute at 56° C., one minute at 72° C.). All amplified products weresequence verified (Yamaguchi et al., J. Cell Biol. 165:275-285, 2004).Control reactions were performed in the absence of reverse transcriptaseand were negative. Each experiment was repeated at least in triplicateindependently.

Immunoblotting

Cultures from 100 mm dishes were solubilized in 500 μl extraction buffercontaining 1% Nonidet P 40 (Calbiochem, San Diego, Calif.), 0.01% SDS,0.1 M Tris:HCl, pH 7.2, and Protease Inhibitor cocktail (Roche,Mannheim, Germany). Protein concentrations of extracts were measuredusing the BCA protein assay kit (Pierce, Rockford, Ill.). Cell extracts(1 μg) were separated on 8-14% gradient SDS polyacrylamide gels(Invitrogen). After electrophoresis, proteins were transferredelectrophoretically from the gels to Immobilon-P transfer membranes(Millipore, Bedford, Mass.). The filters were incubated in the presenceof antibodies to PAR2 (at 1:1,000, Abcam, Cambridge, Mass.), KLEIP (at1:1,000) (Hara et al., Mol. Biol. Cell 15:1172-1184, 2004), β-catenin(at 1:1,000, Cell Signaling Technology, Beverly, Mass.), GSK-3β (at1:1,000, Cell Signaling Technology), pGSK-3β (at 1:1,000, Cell SignalingTechnology), PKCα (at 1:10,000, Sigma, St. Louis, Mo.), PKCβ2 (at1:10,000, Sigma), ERK1/2 (p44/42 MAP Kinase antibody) (at 1:1,000, CellSignaling Technology), pERK1/2 (at 1:1,000, Cell Signaling Technology)or 3-actin (at 1:3,000, AC-15, Abcam) at 23° C. for one hour. They werethen washed and incubated with horseradish peroxidase-linked anti-rabbitor anti-mouse whole antibodies (at 1:1,000, Amersham) at roomtemperature for one hour. Antigens were detected using an ECL-plusWestern Blotting Detection System (Amersham).

Immunocytochemical Staining

Keratinocyte cultures in two well Lab-Tek chamber slides (Nalge NuncInternational Corp., Naperville, Ill.) were processed for indirectimmunofluorescence to detect the expression of signal transductionproteins using primary antibodies to GSK-3β (1:50, Cell SignalingTechnology), phospho-GSK-3β which is specific for GSK-3β phosphorylatedat Ser9 (at 1:100, Cell Signaling Technology), β-catenin (at 1:50, CellSignaling Technology and Santa Cruz, Santa Cruz, Calif.), PKC_(β1) (at1:1,000, Sigma), and PKCα (at 1:1,000, Sigma).

Bound antibodies were visualized with appropriate secondary antibodies,Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (Molecular Probes, Inc.,Eugene, Oreg.) and Alexa Fluor® 594 mouse anti-rabbit IgG (H+L)(Molecular Probes) at 37° C. for 30 minutes at 1:500 dilution with 5%goat serum. DAPI (Vector, Burlingame, Calif.) was used as acounter-stain. The fluorescence of green produced by Alexa 488®, of redproduced by Alexa 594®, and of blue by DAPI was observed and capturedusing a Leica DMR B/D MLD fluorescence microscope (Leica, Wetzlar,Germany) and a Dage-MTI 3CCD 3-chip color video camera (Dage-MTI,Michigan City, Ind.). Confocal microscopy was also used to investigatethe localization, as detailed by (Hoashi et al., J. Biol. Chem.280:14006-14016, 2005).

Example 7 DKK1 Suppresses the Uptake of Melanin and PromotesKeratinocyte Growth and Density

This Example demonstrates the effects of DKK1 on the proliferation ofhuman keratinocytes and their uptake of melanin granules. HaCaTkeratinocytes were mock-transfected or were transfected with DKK1, andtheir uptake of melanin granules purified from MNT1 melanoma cells wasmeasured (FIG. 7). After incubation for 24 hours, keratinocytestransfected with DKK1 phagocytosed significantly less melanin than didthose transfected with a control vector (FIG. 7). Further, transfectionof DKK1 into normal human keratinocytes (FIG. 7) or treatment of thosecells with recombinant human DKK1 (rhDKK1) similarly stimulated theirgrowth and density, as recently reported for human mesenchymal stemcells (Gregory et al., J. Biol. Chem. 280:2309-2323, 2005).

To elucidate DKK1 suppression of the uptake of melanin and promotion ofkeratinocyte growth and density, human keratinocytes were stimulatedwith 50 ng/ml rhDKK1 for two hours, harvested total RNA from those cellsand from untreated controls, and then analyzed those transcripts usingoligonucleotide-DNA microarray technology. Up-regulated ordown-regulated genes are summarized in Tables 7-11. Treatment ofkeratinocytes with DKK1 had varied effects on genes related to cellcontraction, to apoptosis (TNFRSF1A-associated via death domain; TRADD),to non-canonical Wnt signaling pathways, to protein kinase Cβ1 (PKCβ1),to HOX transcription factors (HoxD8 and 12) and to keratins (keratin 9).The differences in mRNA expression levels of interesting genes wereconfirmed using RT-PCR, which validated that levels of some genes (suchas Kelch-like ECT2 interacting protein (KLEIP) and connexin 30 (gapjunction protein, β6; GJB6)) were up-regulated in response to DKK1treatment, whereas DKK1 down-regulated the expression levels of othergenes (such as MITF, procollagen-proline, 2-oxoglutarate 4-dioxygenase(P4HA2), Tubby like protein 3 (Tulp3) and proteinase-activatedreceptor-2 (PAR2; coagulation factor II receptor-like 1; thrombinreceptor-like 1)) (FIG. 8). Although Snrpn expression was one of the top30 genes up-regulated by DKK1, that was not confirmed by RT-PCR; inaddition, no change was detected in BMP21K (GAPDH serves as a loadingcontrol).

TABLE 7 DKK1/NS DKK1 NS Gene Description Top 30 genes up-regulated genesby 2-hour treatment with DKK1* 9.54 4531 475 SUHW4 suppressor of hairywing homolog 4 (Drosophila) (SUHW4), transcript variant 2, mRNA. 8.507572 890 C14orf1 chromosome 14 open reading frame 1 (C14orf1), mRNA.8.12 5994 739 PCSK4 proprotein convertase subtilisin/kexin type 4(PCSK4), mRNA. 7.61 7126 937 EPHA5 EphA5 (EPHA5), transcript variant 2,mRNA. 6.00 3141 524 NRXN1 neurexin 1 (NRXN1), transcript variant β,mRNA. 5.85 8444 1443 DGKH Diacylglycerol kinase, eta 5.55 8480 1527ZNF91 zinc finger protein 91 (HPF7, HTF10) (ZNF91), mRNA. 5.31 2536 478RORB RAR-related orphan receptor B (RORB), mRNA. 4.58 5080 1108 GRIA1Glutamate receptor, ionotropic, AMPA 1 4.21 4162 989 CDCA2 cell divisioncycle associated 2 (CDCA2), mRNA. 3.94 3148 799 ECEL1 endothelinconverting enzyme-like 1 (ECEL1), mRNA. 3.69 5276 1432 FOXG1B forkheadbox G1B (FOXG1B), mRNA. 3.53 2212 626 KLEIP Kelch-like ECT2 interactingprotein 3.35 22237 6646 DNAJC10 DnaJ (Hsp40) homolog, subfamily C,member 10 (DNAJC10), mRNA. 3.32 3430 1033 TRHR thyrotropin-releasinghormone receptor (TRHR), mRNA. 3.25 2129 655 WISP2 WNT1 induciblesignaling pathway protein 2 (WISP2), mRNA. 3.23 2793 865 FBXO31 F-boxprotein 31 3.13 3182 1016 GABRR1 γ-aminobutyric acid (GABA) receptor,rho 1 (GABRR1), mRNA. 3.08 5150 1670 ART3 ADP-ribosyltransferase 3(ART3), mRNA. 3.06 2150 703 SNRPN SNRPN upstream reading frame 3.04 2681881 GTDC1 glycosyltransferase-like 1 (GTDC1), mRNA. 3.01 5032 1669CACNG2 Calcium channel, voltage-dependent, γ subunit 2 3.01 3631 1207DBH dopamine β-hydroxylase (dopamine β-monooxygenase) (DBH), mRNA. 3.012292 763 SLC24A3 solute carrier family 24 (sodium/potassium/calciumexchanger), member 3 (SLC24A3), mRNA. 2.97 2378 800 PRKCB1 proteinkinase C, β1 (PRKCB1), transcript variant 1, mRNA. 2.88 3698 1282 PRSS35protease, serine, 35 (PRSS35), mRNA. 2.81 2329 829 RAB39B RAB39B, memberRAS oncogene family (RAB39B), mRNA. 2.80 3579 1280 ADRB3 adrenergic,β-3-, receptor (ADRB3), mRNA 2.71 8381 3091 SENP6 SUMO1/sentrin specificprotease 6 (SENP6), mRNA. 2.71 4710 1741 NR2C2 nuclear receptorsubfamily 2, group C, member 2 (NR2C2), mRNA. *Genes listed are thosewhose expression levels in DKK1-treated keratinocytes were >2,000(65,000 is the maximum) after 2 hr treatment with DKK1; data arereported as means of 3 independent experiments Top 30 genesDown-regulated genes by 2-hour treatment with DKK1** 0.04 715 19559CSPG3 chondroitin sulfate proteoglycan 3 (neurocan) (CSPG3), mRNA. 0.061094 19633 TULP3 Tubby like protein 3 0.12 3026 25078 LRMPlymphoid-restricted membrane protein (LRMP), mRNA. 0.14 2930 21426CNTNAP2 contactin associated protein-like 2 (CNTNAP2), mRNA. 0.15 355923856 COL3A1 collagen, type III, α1 (Ehlers-Danlos syndrome type IV,autosomal dominant) (COL3A1), mRNA. 0.17 1633 9732 GNAT1 Guaninenucleotide binding protein (G protein), α transducing activitypolypeptide 1 0.17 6119 35544 TRADD TNFRSF1A-associated via death domain(TRADD), transcript variant 1, mRNA. 0.19 8435 44668Anti-streptococcal/anti-myosin immunoglobulin kappa light chain variableregion 0.23 13976 60058 P4HA2 procollagen-proline, 2-oxoglutarate4-dioxygenase (proline 4- hydroxylase), α polypeptide II (P4HA2), mRNA.0.27 2929 10666 IRS4 insulin receptor substrate 4 (IRS4), mRNA. 0.295207 18020 MAPK9 mitogen-activated protein kinase 9 (MAPK9), transcriptvariant 2, mRNA. 0.29 14354 49168 PTCH2 patched homolog 2 (Drosophila)(PTCH2), mRNA. 0.32 7783 24293 PDXP pyridoxal (pyridoxine, vitamin B6)phosphatase (PDXP), mRNA. 0.33 6206 18788 hSyn brain synembryn (hSyn),mRNA. 0.35 1920 5551 TARSH target of Nesh-SH3 (TARSH), mRNA. 0.35 964627748 MAN1C1 mannosidase, α, class 1C, member 1 (MAN1C1), mRNA. 0.3518040 51769 RAB25 RAB25, member RAS oncogene family (RAB25), mRNA. 0.351109 3133 GAD1 glutamate decarboxylase 1 (brain, 67 kDa) (GAD1),transcript variant GAD67, mRNA. 0.36 12314 34502 SERPINH1 serine (orcysteine) proteinase inhibitor, clade H (HSP 47), member 1, (collagenbinding protein 1) (SERPINH1), mRNA. 0.36 1973 5455 RPS4X Ribosomalprotein S4, X-linked 0.36 15682 43145 RPS4Y1 ribosomal protein S4,Y-linked 1 (RPS4Y1), mRNA. 0.37 22985 62579 RHOBTB2 Rho-related BTBdomain containing 2 (RHOBTB2), mRNA. 0.37 16119 43756 MITFmicrophthalmia-associated transcription factor (MITF), transcriptvariant 5, mRNA. 0.38 4534 12021 KISS1 KiSS-1 metastasis-suppressor(KISS1), mRNA. 0.38 772 2021 ACTL6B actin-like 6B (ACTL6B), mRNA. 0.394267 11042 PHLDB3 Pleckstrin homology-like domain, family B, member 30.39 18262 46894 XPA xeroderma pigmentosum, complementation group A(XPA), mRNA. 0.39 4619 11830 GTF2A1 General transcription factor IIA, 1,19/37 kDa 0.39 6012 15390 UMP-CMPK UMP-CMP kinase (UMP-CMPK), mRNA. 0.391696 4327 JRKL jerky homolog-like (mouse) (JRKL), mRNA. **Genes listedare those whose expression levels in mock-treated keratinocyteswere >2,000 (65,000 is the maximum) after 2 hr treatment with DKK1; dataare reported as means of 3 independent experiments.

TABLE 8 DKK1/NS DKK1 NS Gene Description Receptor-Related GenesUp-regulated by Treatment with DKK1 for 2 hr* 5.31 2536 478 RORBRAR-related orphan receptor B (RORB), mRNA. 4.58 5080 1108 GRIA1Glutamate receptor, ionotropic, AMPA 1 3.32 3430 1033 TRHRthyrotropin-releasing hormone receptor (TRHR), mRNA. 3.13 3182 1016GABRR1 γ-aminobutyric acid (GABA) receptor, rho 1 (GABRR1), mRNA. 2.803579 1280 ADRB3 adrenergic, β-3-, receptor (ADRB3), mRNA. 2.75 2076 755EPHA6 EPH receptor A6 2.71 4710 1741 NR2C2 nuclear receptor subfamily 2,group C, member 2 (NR2C2), mRNA. 2.61 2495 956 FCER1A Fc fragment ofIgE, high affinity I, receptor for; α polypeptide 2.60 40698 15632 SORL1sortilin-related receptor, L(DLR class) A repeats-containing (SORL1),mRNA. 2.47 2291 926 PTGER3 Prostaglandin E receptor 3 (subtype EP3) 2.452034 829 TNFRSF17 tumor necrosis factor receptor superfamily, member 17(TNFRSF17), mRNA. 2.40 1166 486 GRIN3A glutamate receptor, ionotropic,N-methyl-D-aspartate 3A (GRIN3A), mRNA. 2.37 4562 1921 P2RY1 purinergicreceptor P2Y, G-protein coupled, 1 (P2RY1), mRNA. 2.36 1715 726 PPFIA2protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF),interacting protein (liprin), α2 (PPFIA2), mRNA. 2.35 1041 443 ITGA4integrin, α4 (antigen CD49D, α4 subunit of VLA-4 receptor) (ITGA4),mRNA. 2.35 3671 1565 NCR2 natural cytotoxicity triggering receptor 2(NCR2), mRNA. 2.33 1397 599 CHRM3 Cholinergic receptor, muscarinic 32.21 3113 1412 GPR37 G protein-coupled receptor 37 (endothelin receptortype B-like) (GPR37), mRNA. 2.20 4655 2113 VPS16 Protein tyrosinephosphatase, receptor type, A 2.20 1990 904 LRP4 Low density lipoproteinreceptor-related protein 4 2.19 2528 1155 PTPRK Protein tyrosinephosphatase, receptor type, K 2.19 1702 778 PTPRK Protein tyrosinephosphatase, receptor type, K 2.17 1147 530 NR2E1 nuclear receptorsubfamily 2, group E, member 1 (NR2E1), mRNA. 2.12 2743 1297 P2RY10purinergic receptor P2Y, G-protein coupled, 10 (P2RY10), transcriptvariant 2, mRNA. 2.11 1195 567 TFR2 transferrin receptor 2 (TFR2), mRNA.2.11 2486 1181 NR3C2 nuclear receptor subfamily 3, group C, member 2(NR3C2), mRNA. 2.10 1337 635 EDG6 endothelial differentiation,G-protein-coupled receptor 6 (EDG6), mRNA. 2.10 1907 908 TRPM8 transientreceptor potential cation channel, subfamily M, member 8 (TRPM8), mRNA.2.06 3880 1885 ILT7 leukocyte immunoglobulin-like receptor, subfamily A(without TM domain), member 4 (ILT7), mRNA. 2.05 2298 1120 PVRL3Poliovirus receptor-related 3 2.05 1219 595 NR4A2 nuclear receptorsubfamily 4, group A, member 2 (NR4A2), transcript variant 4, mRNA. 2.043027 1486 HRH3 histamine receptor H3 (HRH3), mRNA. 2.04 1975 970 GPR23 Gprotein-coupled receptor 23 (GPR23), mRNA 2.00 1075 537 ADRB1adrenergic, β-1-, receptor 2.00 11326 5670 MST1R macrophage stimulating1 receptor (c-met-related tyrosine kinase) (MST1R), mRNA *Genes listedare those whose expression levels in DKK1-treated keratinocyteswere >1,000 (65,000 is the maximum) after 2 hr treatment with DKK1; datafor genes with DKK1/NS ratio >2.00 are reported as means of 3independent experiments. KREMEN1(kringle containing transmembraneprotein 1), transcript variant 2, was 3.5-797-227. Receptor-RelatedGenes Down-regulated by Treatment with DKK1 for 2 hr** 0.16 321 1976GRIN3A glutamate receptor, ionotropic, N-methyl-D-aspartate 3A (GRIN3A),mRNA. 0.17 6119 35544 TRADD TNFRSF1A-associated via death domain(TRADD), transcript variant 1, mRNA. 0.18 182 1000 OR12D3 olfactoryreceptor, family 12, subfamily D, member 3 (OR12D3), mRNA. 0.24 343 1432IL10RA interleukin 10 receptor, α (IL10RA), mRNA. 0.27 2929 10666 IRS4insulin receptor substrate 4 (IRS4), mRNA. 0.35 602 1701 GRM6 glutamatereceptor, metabotropic 6 (GRM6), mRNA. 0.36 551 1520 GRK4 Gprotein-coupled receptor kinase 4 0.36 719 1978 MS4A2 membrane-spanning4-domains, subfamily A, member 2 (Fc fragment of IgE, high affinity I,receptor for β polypeptide) (MS4A2) mRNA. 0.40 640 1592 LOC219347Similar to nuclear DNA-binding protein; small unique nuclear receptorcorepressor; C1D DNA-binding protein 0.41 439 1075 GPR116 Gprotein-coupled receptor 116 (GPR116), mRNA. 0.42 1959 4652 PTPRSprotein tyrosine phosphatase, receptor type, S (PTPRS), transcriptvariant 3, mRNA. 0.43 4032 9335 LTBR lymphotoxin β receptor (TNFRsuperfamily, member 3) (LTBR), mRNA. 0.45 662 1486 AGTR2 angiotensin IIreceptor, type 2 (AGTR2), mRNA. 0.45 757 1691 GNRHRGonadotropin-releasing hormone receptor (GNRHR), mRNA. 0.47 1568 3312OR5V1 olfactory receptor, family 5, subfamily V, member 1 (OR5V1), mRNA.0.48 4914 10334 ANTXR2 anthrax toxin receptor 2 (ANTXR2), mRNA. **Geneslisted are those whose expression levels in mock-treated keratinocyteswere >1,000 (65,000 is the maximum) after 2 hr treatment with DKK1; datafor genes with DKK1/NS ratio <0.50 are reported as means of 3independent experiments.

TABLE 9 DKK1/NS DKK1 NS Gene Description Wnt-Related Genes Up-regulatedby Treatment with DKK1 for 2 hr* 3.60 1449 403 APC2 adenomatosispolyposis coli 2 (APC2), mRNA. 3.51 797 227 KREMEN1 kringle containingtransmembrane protein 1 (KREMEN1), transcript variant 2, mRNA. 3.25 2129655 WISP2 WNT1 inducible signaling pathway protein 2 (WISP2), mRNA. 2.972378 800 PRKCB1 protein kinase C, β1 (PRKCB1), transcript variant 1,mRNA. 2.56 1696 664 PPP2R2B Protein phosphatase 2 (formerly 2A),regulatory subunit B (PR 52), β isoform 2.50 1081 431 NFATC2 Nuclearfactor of activated T-cells, cytoplasmic, calcineurin-dependent 2 2.213811 1721 PRKCH Protein kinase C, eta 1.76 1849 1048 WNT9B wingless-typeMMTV integration site family, member 9B (WNT9B), mRNA. 1.74 1384 797PRKCG protein kinase C, γ (PRKCG), mRNA. 1.70 2116 1245 SFRP4 secretedfrizzled-related protein 4 (SFRP4), mRNA. 1.65 7275 4397 WISP1 WNT1inducible signaling pathway protein 1 (WISP1), transcript variant 1,mRNA. Wnt-Related Genes Down-regulated by Treatment with DKK1 for 2 hr**1.65 1826 1105 FRAT1 frequently rearranged in advanced T-cell lymphomas(FRAT1), transcript variant 1, mRNA. 1.64 6852 4184 DVL2 dishevelled,dsh homolog 2 (Drosophila) (DVL2), mRNA. 1.61 2132 1321 PRKCA proteinkinase C, α (PRKCA), mRNA. 1.60 10952 6829 DAAM1 Dishevelled associatedactivator of morphogenesis 1 1.59 2605 1642 EP300 E1A binding proteinp300 (EP300), mRNA. 1.58 8442 5334 SMAD2 SMAD, mothers against DPPhomolog 2 (Drosophila) (SMAD2), transcript variant 1, mRNA. 1.57 70394474 PPP3CB protein phosphatase 3 (formerly 2B), catalytic subunit, βisoform (calcineurin A β) (PPP3CB), mRNA. 1.57 1787 1139 TCF7transcription factor 7 (T-cell specific, HMG-box) (TCF7), transcriptvariant 2, mRNA. 1.54 1412 915 SIP Siah-interacting protein (SIP), mRNA.1.53 5306 3469 PRKCH protein kinase C, eta (PRKCH), mRNA. 1.45 2468 1706TCF7L2 transcription factor 7-like 2 (T-cell specific, HMG-box)(TCF7L2), mRNA. 0.29 5207 18020 MAPK9 mitogen-activated protein kinase 9(MAPK9), transcript variant 2, mRNA. 0.41 861 2098 PRKACB proteinkinase, cAMP-dependent, catalytic, β (PRKACB), transcript variant 3,mRNA. 0.46 514 1114 CAMK2A calcium/calmodulin-dependent protein kinase(CaM kinase) II α (CAMK2A), transcript variant 2, mRNA. 0.46 1153 2491NFATC3 nuclear factor of activated T-cells, cytoplasmic,calcineurin-dependent 3 (NFATC3), transcript variant 2, mRNA. 0.48 8131700 TBL1X transducin (β)-like 1X-linked (TBL1X), mRNA. 0.52 4010 7706CSNK1G2 Casein kinase 1, γ2 (CSNK1G2), mRNA. 0.54 787 1454 SFRP5secreted frizzled-related protein 5 (SFRP5), mRNA. 0.55 689 1257 WNT2wingless-type MMTV integration site family member 2 (WNT2), mRNA. 0.551411 2570 NFATC1 nuclear factor of activated T-cells, cytoplasmic,calcineurin-dependent 1 (NFATC1), transcript variant 4, mRNA. 0.57 1242021611 CSNK1A1 Casein kinase 1, α1 (CSNK1A1), mRNA. 0.58 2849 4888 PPARDperoxisome proliferative activated receptor, δ (PPARD), transcriptvariant 1, mRNA. 0.59 640 1091 WNT1 wingless-type MMTV integration sitefamily, member 1 (WNT1), mRNA. 0.60 635 1051 NFATC2 nuclear factor ofactivated T-cells, cytoplasmic, calcineurin-dependent 2 (NFATC2),transcript variant 1, mRNA. 0.61 2908 4791 TLE1 Transducin-like enhancerof split 1 (E(sp1) homolog, Drosophila) 0.61 5749 9400 SHFM3 Splithand/foot malformation (ectrodactyly) type 3 0.62 8840 14278 SKP1AS-phase kinase-associated protein 1A (p19A) (SKP1A), transcript variant2, mRNA. 0.62 18626 29873 DKK1 dickkopf homolog 1 (Xenopus laevis)(DKK1), mRNA. 0.63 650 1035 SMAD3 SMAD, mothers against DPP homolog 3(Drosophila) 0.63 662 1051 SKP1A S-phase kinase-associated protein 1A(p19A) (SKP1A), transcript variant 1, mRNA. 0.63 920 1459 SHFM3 Splithand/foot malformation (ectrodactyly) type 3 0.64 1463 2296 CAMK2Bcalcium/calmodulin-dependent protein kinase (CaM kinase) II β (CAMK2B),transcript variant 8, mRNA. 0.65 1129 1739 PRKACB protein kinase,cAMP-dependent, catalytic, β (PRKACB), transcript variant 1, mRNA. 0.6517207 26320 RBX1 ring-box 1 (RBX1), mRNA. 0.66 832 1253 PRKCE Proteinkinase C, epsilon 0.67 19582 29362 PRICKLE1 prickle-like 1 (Drosophila)(PRICKLE1), mRNA. 0.67 23176 34546 YY1 YY1 transcription factor (YY1),mRNA. 0.67 29791 44369 AES Amino-terminal enhancer of split 0.68 44186542 AXIN1 axin 1 (AXIN1), transcript variant 2, mRNA. 0.68 7298 10757SLC9A3R1 Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3regulator 1 (SLC9A3R1), mRNA. 0.68 7036 10320 TBL1XR1 transducin(β)-like 1X-linked receptor 1 (TBL1XR1), mRNA. 0.68 8962 13140 RUVBL1RuvB-like 1 (E. coli) (RUVBL1), mRNA. 0.68 3428 5024 JUN v-jun sarcomavirus 17 oncogene homolog (avian) (JUN), mRNA. 0.69 2743 3989 PKN2protein kinase N2 (PKN2), mRNA. 0.69 14324 20809 CSNK1D Casein kinase 1,δ (CSNK1D), transcript variant 1, mRNA. 0.70 2005 2879 PYGO2 pygopus 2(PYGO2), mRNA. 0.70 6558 9373 NOTCH1 Notch homolog 1,translocation-associated (Drosophila) (NOTCH1), mRNA. 0.70 2357 3365SOX17 SRY (sex determining region Y)-box 17 (SOX17), mRNA. **Geneslisted are those whose expression levels in mock-treated keratinocyteswere >1,000 (65,000 is the maximum) after 2 hr treatment with DKK1; datafor genes with DKK1/NS ratio <0.70 are reported as means of 3independent experiments.

TABLE 10 DKK1/NS DKK1 NS Gene Description HOX-Related Genes Up-regulatedby Treatment with DKK1 for 2 hr* 3.49 1986 569 DLX6 PREDICTED: Homosapiens distal-less homeo box 6 (DLX6), mRNA. 3.04 325 107 SHOX shortstature homeobox (SHOX), transcript variant SHOXa, mRNA. 2.62 1520 580HOXB13 homeo box B13 (HOXB13), mRNA. 2.37 2125 895 LOC135935 PREDICTED:Homo sapiens similar to OG-2 homeodomain protein-like; similar to U65067(PID: g1575526) (LOC135935), mRNA. 1.94 5511 2836 PROX1 prospero-relatedhomeobox 1 (PROX1), mRNA. 1.74 5145 2961 PITX2 paired-like homeodomaintranscription factor 2 (PITX2), transcript variant 1, mRNA. 1.72 1578915 HOXB1 homeo box B1 (HOXB1), mRNA. 1.70 2776 1636 HOXB9 homeo box B9(HOXB9), mRNA. 1.67 15659 9358 LHX6 LIM homeobox 6 (LHX6), transcriptvariant 2, mRNA. 1.67 1605 962 HOXC4 homeo box C4 (HOXC4), transcriptvariant 1, mRNA. 1.65 1635 990 GBX2 gastrulation brain homeo box 2(GBX2), mRNA. 1.62 1341 827 HOXA13 homeo box A13 (HOXA13), mRNA. 1.541231 799 CART1 cartilage paired-class homeoprotein 1 (CART1), mRNA. 1.524364 2873 HOXA4 homeo box A4 (HOXA4), mRNA. 1.50 2705 1805 HLXB9 homeobox HB9 (HLXB9), mRNA. 1.48 1585 1074 CDX4 caudal type homeo boxtranscription factor 4 (CDX4), mRNA. 1.47 7675 5218 PKNOX1 PBX/knotted 1homeobox 1 (PKNOX1), transcript variant 1, mRNA. *Genes listed are thosewhose expression levels in DKK1-treated keratinocytes were >1,000(65,000 is the maximum) after 2 hr treatment with DKK1; data for geneswith DKK1/NS ratio >1.45 are reported as means of 3 independentexperiments. HOX-Related Genes Down-regulated by Treatment with DKK1 for2 hr** 0.35 406 1171 BAPX1 bagpipe homeobox homolog 1 (Drosophila)(BAPX1), mRNA. 0.42 1044 2501 HOXC5 homeo box C5 (HOXC5), mRNA. 0.484480 9420 PHC2 polyhomeotic-like 2 (Drosophila) (PHC2), transcriptvariant 2, mRNA. 0.48 856 1785 LMX1B LIM homeobox transcription factor1, β (LMX1B), mRNA. 0.48 1033 2152 HOXB8 homeo box B8 (HOXB8), mRNA.0.50 1086 2163 HMX1 homeo box (H6 family) 1 (HMX1), mRNA. 0.53 611011499 PKNOX2 PBX/knotted 1 homeobox 2 (PKNOX2), mRNA. 0.53 1030 1938BARX2 BarH-like homeobox 2 (BARX2), mRNA. 0.55 3371 6164 ZFHX1B zincfinger homeobox 1b (ZFHX1B), mRNA. 0.55 1137 2052 HOXD8 homeo box D8(HOXD8), mRNA. 0.56 4305 7660 HOXA9 homeo box A9 (HOXA9), transcriptvariant 2, mRNA. 0.57 818 1442 DUX1 double homeobox, 1 (DUX1), mRNA.0.60 6069 10151 HIPK4 homeodomain interacting protein kinase 4 (HIPK4),mRNA. 0.61 1324 2171 SIX3 sine oculis homeobox homolog 3 (Drosophila)(SIX3), mRNA. 0.62 2695 4359 ZHX2 zinc fingers and homeoboxes 2 (ZHX2),mRNA. 0.62 730 1176 LOC360030 Homeobox C14 0.62 2611 4194 MSX2 Msh homeobox homolog 2 (Drosophila) 0.63 1659 2641 ZFHX2 PREDICTED: Homo sapienszinc finger homeobox 2 (ZFHX2), mRNA. 0.63 2866 4538 HOXA11 homeo boxA11 (HOXA11), mRNA. 0.64 1013 1580 HOXB5 homeo box B5 (HOXB5), mRNA.0.65 1466 2263 VENTX2P1 VENT-like homeobox 2 pseudogene 1 (VENTX2P1) onchromosome X. 0.67 3704 5533 MEOX1 Mesenchyme homeo box 1 0.68 980 1451HOXD11 homeo box D11 (HOXD11), mRNA. 0.69 1568 2267 HOXD4 Homeo box D40.69 902 1304 IPF1 insulin promoter factor 1, homeodomain transcriptionfactor (IPF1), mRNA. 0.70 2763 3924 ZHX3 zinc fingers and homeoboxes 3(ZHX3), mRNA. **Genes listed are those whose expression levels inmock-treated keratinocytes were >1,000 (65,000 is the maximum) after 2hr treatment with DKK1; data for genes with DKK1/NS ratio <0.70 arereported as means of 3 independent experiments.

TABLE 11 Keratin-Related Genes Up-regulated by Treatment with DKK1 for 2hr* DKK1/NS DKK1 NS Gene Description 2.10 2531 1205 KRT9 keratin 9(epidermolytic palmoplantar keratoderma) (KRT9), mRNA. 1.74 11545 6627KRTHB2 keratin, hair, basic, 2 (KRTHB2), mRNA. 1.47 3537 2401 KRT20keratin 20 (KRT20), mRNA. 1.46 65000 44518 KRT16 keratin 16 (focalnon-epidermolytic palmoplantar keratoderma) (KRT16), mRNA. 0.40 857 2168KRTAP3-2 keratin associated protein 3-2 (KRTAP3-2), mRNA. 0.50 1870 3725KRTAP4-14 keratin associated protein 4-14 (KRTAP4-14), mRNA. 0.58 1107719009 KRT1 keratin 1 (epidermolytic hyperkeratosis) (KRT1), mRNA. 0.586453 11059 KRTAP2.1A Keratin associated protein KRTAP2.1A 0.59 1598 2723KRTAP4-8 Keratin associated protein 4-8 0.62 10754 17429 K6HFcytokeratin type II (K6HF), mRNA. 0.62 7534 12104 KRT4 keratin 4 (KRT4),mRNA. 0.68 946 1395 KRTAP1-3 keratin associated protein 1-3 (KRTAP1-3),mRNA. 0.68 965 1417 HUMCYT2A cytokeratin 2 (HUMCYT2A), mRNA. 0.68 1297619027 KRT13 keratin 13 (KRT13), transcript variant 2, mRNA. 0.70 25563648 KRTAP2-4 keratin associated protein 2-4 (KRTAP2-4), mRNA. **Geneslisted are those whose expression levels in mock-treated keratinocyteswere >1,000 (65,000 is the maximum) after 2 hr treatment with DKK1; datafor genes with DKK1/NS ratio <0.70 are reported as means of 3independent experiments.

Example 8 Expression Patterns of Proteins Related to the Wnt/β-CateninSignaling Pathway

This Example illustrates the expression patterns of PAR2, KLEIP andvarious proteins related with the Wnt/β-catenin signaling pathway.Extracts of keratinocytes were harvested after two hours of treatmentwith 50 ng/ml rhDKK1 and at three days after transfection with DKK1, andthey were analyzed using Western blotting (similar results were found inboth protocols, extracts at 3 days after transfection with DKK1 areshown in FIG. 9). For KLEIP, three non-specific bands were found inkeratinocytes, as previously reported (Hara et al., Mol. Biol. Cell15:1172-1184, 2004), but the correct band was also detected (at 64 kDa,arrow) for KLEIP whose expression level in keratinocytes was increasedin response to DKK1. No recognizable signals for PAR2 were detectable ateither time point.

The expression levels of nuclear and cytoplasmic β-catenin weredown-regulated after two hours and five days of treatment with DKK1.GSK3β is not only an enzyme involved in the control of glycogenmetabolism, but it also regulates a variety of cellular functionsincluding Wnt signaling pathways (Cohen & Goedert, Nature Rev. DrugDiscov. 3:479-487, 2004). Inactivation of GSK3β via phosphorylation ofSer9 is the major route by which insulin activates muscle glycogensynthase (McManus et al., EMBO J. 24:1571-1583, 2005). The expression ofGSK3β phosphorylated at Ser9 was investigated, and it was found that itwas up-regulated in response to DKK1 at both time points, althoughoverall GSK3β expression was similar at both time points.

Since PKCα is reported to inactivate GSK3β (Fang et al., Mol. Cell.Biol. 22:2099-2100, 2002), the expression of PKC isoforms was examined.Treatment with DKK1 up-regulated the expression of PKCα and PKCβ2 atboth time points. Extracellular-signal regulated kinase (ERK) 1/2 playsimportant roles in keratinocyte proliferation and migration (He et al.,J. Biol. Chem. 279:53867-53874, 2004; Pullar et al., FASEB J. 20:76-86,2006). Although levels of ERK were not changed by DKK1, expression ofthe phosphorylated form of ERK1/2 (at Thr202/Tyr204) was up-regulated inresponse to DKK1. β-actin serves as a loading control. Finally, theseexpression patterns were validated by immunocytochemistry. Theexpression of β-catenin was decreased in response to DKK1, whereas thatof PKCα and PKCβ1 was increased at 2 hours and 5 days after DKK1treatment (FIGS. 10 and 11).

The expression patterns of several key keratinocyte proteins identifiedin the microarray analysis were also studied in vivo usingimmunohistochemistry in palm and sole skin as compared tononpalmoplantar trunk skin. The expression of β-catenin and PAR2 wasdecreased in skin on the palms and soles whereas keratin 9 was onlyobserved in palmoplantar skin (FIG. 12).

Example 9 DDK1 Treatment Increased Skin Thickness and Reduced MelaninContent

This example demonstrates that the secretion of DDK1 by fibroblasts wasresponsible for the physiological differences observed betweenpalmoplantar skin and nonpalmoplantar skin. Reconstructed skins weregrown in the presence or absence of rhDKK1 (FIG. 13). After 7 days, therhDDK1-treated skins were already significantly less pigmented than themock-treated controls. The difference was clearer after 7 or 10 days oftreatment (FIG. 13A). This difference in pigmentation was confirmed byFontana-Masson staining which showed a marked decrease in melanincontent in rhDKK1-treated skins (FIG. 7B). The skin, and particularlythe stratum corneum, were significantly thicker after treatment byrhDDK1 (FIG. 7C).

The immunostaining of these reconstructed skins was consistent with thefindings of skin from normal volunteers (as noted above), which revealeddecreased expression of PAR2 and β-catenin after 10 days of treatmentwith rhDDK1 (FIG. 14). Several cells positive for keratin 9 alsoappeared after 10 days of treatment with rhDKK1.

Example 10 Summary of Regulation of Skin Thickness, by DKK1

DKK1, which is secreted at high levels by fibroblasts in palmoplantarskin, decreases melanin uptake by keratinocytes and increases theirproliferation. Physiologically, keratinocytes in hypopigmentedpalmoplantar epidermis are more proliferative (generating a much thickerskin) but at the same time are less pigmented. As demonstrated herein,DKK1 decreases melanin uptake by keratinocytes and increases theirproliferation. DNA microarray analysis was used to elucidate theseeffects, and to compare gene expression patterns of keratinocytestreated or untreated with DKK1.

Regulation of Proliferation and Cell Density

Tulp3, which is ubiquitously expressed throughout embryonic development,belongs to the tubby-like protein family. Tulp3-knockout mice exhibitembryonic lethality with a failure in neural tube closure characterizedby neuroepithelial apoptosis (Ikeda et al., Hum Mol Genet. 10:1325-1334,2001). The earliest phenotype of Tulp3-knockout mice is a significantreduction in the number of βIII-tubulin-positive neurons in thehindbrain, which suggests that Tulp3 maintains normally differentiatingneuronal cell populations. Apoptosis is restricted to the ventral regionof the neuroepithelium in the hindbrain of Tulp3-knockout mice,suggesting that Tulp3 is involved in selective cell death in specifictypes of cells (Ikeda et al., Hum Mol Genet. 10:1325-1334, 2001; Ikedaet al., J Cell Sci 115:9-14, 2002; Carroll et al., Nat Rev Mol Cell Biol5:55-63, 2004). DKK1 down-regulated the expression of Tulp3 inkeratinocytes, which indicates that Tulp3 down-regulates the apoptosisof keratinocytes through βIII-tubulin interactions.

KLEIP is a protein that is involved in actin assembly at sites ofcell/cell adhesion (Hara et al., Mol. Biol. Cell 15:1172-1184, 2004) andwhich associates with ECT2, a Rho nucleotide exchange factor involved incytokinesis (Miki et al., Nature 362:462-465, 1993; Tatsumoto et al., J.Cell Biol. 147:921-928, 1999). Enhanced mRNA and protein expression ofKLEIP by DKK1 may account, at least in part, for the enhanced actinassembly and cytokinesis of keratinocytes in palmoplantar skin, which isconsistent with their increased proliferation and density in thattissue. DKK1 also enhanced the expression level of keratin 9, which canbe induced in non-palmoplantar keratinocytes by palmoplantar fibroblasts(Yamaguchi et al., J. Invest. Dermatol. 112:483-488, 1999), whichindicates that DKK1 can alter keratin dimerization and patterning inkeratinocytes. The altered keratin patterning seen in palmoplantarkeratinocytes due to secreted DKK1 also may account for the thickphenotype of palmoplantar epidermis. Further, a slight up-regulation ofGJB6 was observed at the mRNA level, which suggests that alteredcell/cell communications mediated by gap junctions may also play rolesin regulating the proliferation, migration and differentiation ofkeratinocytes (Brandner et al., J. Invest. Dermatol. 122:1310-1320,2004; Di et al., J. Cell Sci. 118:1505-1514, 2005).

In addition to demonstrating the increased expression of KLEIP inresponse to DKK1 at the mRNA and protein levels, the finding of thedecreased expression of cytoplasmic β-catenin (Yamaguchi et al., J. CellBiol. 165:275-285, 2004) may result in decreased cell/cell contact,which suggests that numerous signals are involved in this pathway.Increased expression of PKCα and PKClβs helps explain the increased celldensity since PKC isozymes regulate the proliferation anddifferentiation of keratinocytes (Papp et al., Exp Dermatol 12:811-824,2003). ERK phosphorylation is activated at the healing wound edge.Additionally, 132-adrenergic receptor, whose activation delays woundhealing, prevents localization of phospho-ERK to the lamellipodial edge(Pullar et al., FASEB J. 20:76-86, 2006). ERK activation in response toDKK1 may also play a role in the stimulation of keratinocyteproliferation and the increased cellular density since ERK is also animportant suppressive regulator of apoptosis (He et al., J Biol Chem279:53867-53874, 2004). Canonical Wnt signals activate β-cateninexpression through the inhibition of β-catenin degradation by multipleprotein complexes including GSK3β, axin and APC. However, Wnt-5ainhibits the canonical Wnt pathway by promoting GSK3β-independentβ-catenin degradation (Topol et al., J. Cell Biol. 162:899-908, 2003).GSK3β is a unique protein in that it is inactivated throughphosphorylation (Cohen and Frame, Nature Rev: Mol. Cell Biol. 2:769-776,2001). Although DKK1 did not affect the overall expression level ofGSK3β, the finding of the elevated phosphorylation of GSK3β at Ser9 inresponse to DKK1 supports a novel pathway for DKK1/Wnt/β-cateninsignaling.

P4HA2, prolyl-4-hydroxylase α-subunit (α2), plays roles in collagenfiber formation, probably in response to hypoxia-inducible transcriptionfactor 1 (Hofbauer et al., Eur. J. Biochem. 270:4515-4522, 2003; Mackayet al., Oncogene 22:2680-2688, 2003; Jarzab et al., Cancer Res65:1587-1597, 2005). DKK1 down-regulates the expression of P4HA2 inkeratinocytes, hence the appearance of the dermis in palmoplantar areasmight also differ from non-palmoplantar areas due to the down-regulatedexpression of this hydroxylase.

Regulation of Pigmentation Production and Distribution DKK1significantly inhibits the uptake of melanin granules by keratinocytes,and thereby decreases pigment in the epithelium. PAR2 is expressed onkeratinocytes and is involved in melanin uptake via phagocytosis(Seiberg et al., Exp. Cell Res. 254:25-32, 2000) in a Rho-dependentfashion (Scott et al., J Invest Dermatol 121:529-541, 2003). PAR2 playsa significant role in modulating pigmentation in a skin type-dependentmanner (Babiarz-Magee et al., Pigment Cell Res. 17:241-251, 2004) and inresponse to UV (Scott et al., J. Invest. Dermatol. 117:1412-1420, 2001).Thus, the decreased melanin uptake elicited in keratinocytes by DKK1 mayin large part be mediated through the suppressed expression of PAR2.DKK1 might also decrease the secretion of melanosomes by melanocytes.

Although detectable levels of MITF protein were not observed inkeratinocytes, differential display and RT-PCR showed that DKK1suppresses the expression of MITF mRNA in those cells. Thus,keratinocytes express MITF at the mRNA level but do not make detectablelevels of MITF protein. However, this result indicates that not onlydoes DKK1 suppress MITF expression in melanocytes (Garraway et al.,Nature 436:117-122., 2005) but also in keratinocytes. The decreaseduptake of melanin granules by keratinocytes elicited by DKK1 issignificant.

Physiological Impact of DKK1

Several key proteins regulated by DDK1 in vitro showed the sameexpression patterns between palmoplantar epidermis and dorsal epidermisin vivo. Indeed, the expression levels of PAR2 and β-catenin were lessin palmoplantar skin compared to dorsal epidermis. In contrast, keratin9 was found in all suprabasal layers of palmoplantar epidermis and wasalmost absent in dorsal skin. These patterns of protein expression werereproduced in reconstructed epidermis (MelanoDerm®) consisting ofcultured human melanocytes and keratinocytes after 10 days of treatmentwith rhDKK1. Not only the expression of PAR2 and β-catenin weredecreased but several cells stained positively for keratin 9 in therhDKK1-treated skin substitutes. Keratin 9 is normally observed only inpalmoplantar epidermis (Knapp et al., J. Cell Biol. 103:657-667, 1986)and its defective function is a cause of epidermolytic palmoplantarkeratoderma (Langbein et al., Differentiation 55:57-71, 1993; Kobayashiet al., FEBS Lett. 386:149-155, 1996). The rhDKK1-treated epidermis alsohad less melanin and a thicker stratum corneum and this tendency tothicken became more significant as the treatment time increased. Thesechanges were so dramatic that they were visually detectable after 7 daysof treatment, and were obvious after 10 days. Although a number of othercells and interactions no doubt also contribute to the regulation ofskin morphology and pigmentation, the fact that DKK1 can recapitulatethe palmoplantar phenotype in the reconstituted skin model emphasizesits key role in those processes. Thus, DKK1 by itself was sufficient toinduce a palmoplantar phenotype in a reconstructed epidermis.

CONCLUSIONS

Numerous up-regulated and down-regulated receptors and HOX-related geneswere found that responded to DKK1, indicating that DKK1 can serve as aligand (agonist) or an antagonist for those receptors and can affect thetopographical/site-specific/anatomically specific distribution of HOX inthe human body. Taken together, DKK1 has various effects on keratinocytegrowth and function, including the up-regulation of cell density and thedecreased melanin uptake, probably through numerous effects on geneexpression patterns.

In conclusion, these findings elucidate why palmoplantar epidermis isthicker and paler than non-palmoplantar epidermis. In addition todermal/epidermal interactions, which play important roles in regulatingkeratinocyte growth and differentiation through numerous growth factors(Szabowski et al., Cell 103:745-755, 2000), the necessity of thosetopographical/site-specific regulations have been proposed (Yamaguchi etal., J. Dermatol. Sci. 40:1-9, 2005). DKK1, a secretory protein highlyexpressed by palmoplantar fibroblasts (Yamaguchi et al., J. Cell Biol.165:275-285, 2004) has various effects on keratinocytes, the major typeof cell in the epidermis. Enhanced KLEIP expression in response to DKK1may play a role in the enhanced cell contraction (compact cellularorganization) in palmoplantar keratinocytes stimulated with DKK1.Additionally, the decreased expression of β-catenin in the cytoplasm ofkeratinocytes that is elicited by DKK1 may result in the loose cell/cellcontact and the increased cellular density seen in palmoplantarepidermis, in which suprabasal keratin 9 expression is also seen (Knappet al., J. Cell Biol. 103:657-667, 1986). PKC isozymes and GSK3β mayparticipate in DKK1/Wnt/β-catenin signaling pathways. The fact that DKK1decreases melanocyte function and proliferation through MITF (Yamaguchiet al., J. Cell Biol. 165:275-285, 2004) and that melanosome transfer isdecreased in keratinocytes in response to DKK1 (probably through PAR2)explains the hypopigmentation seen in the skin on palms and soles.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. A method for treating a dermatological pathology, comprising:topically applying an effective amount of DKK1 to non-palmoplantar skin,thereby inducing the non-palmoplantar skin to develop a palmoplantarphenotype and treating the dermatological pathology, wherein thedermatological pathology is a skin graft, an ulcer, an abrasion, aninjury caused by a repetitive impact or mechanical stress, age-relatedskin changes, skin damage due to steroid treatment, uneven skinpigmentation, hyperpigmentation, post-inflammatory pigmentation,fragrance dermatitis, ephelides, vitiligo, a pigmented birthmark,lentigos, or skin changes due to lichen simplex chronicus, melasma,Addison's disease, Peutz-Jeghers syndrome, acanthosis nigricans,hirsutism, congenital adrenal hyperplasia, polycystic ovarian syndrome,hypertrichosis, porphyria cutanea tarda, anorexia nervosa, or melanoma.2. The method of claim 1, wherein the method increases skin thickness.3. The method of claim 1, wherein the method is an in vitro method. 4.The method of claim 2, wherein the DKK1 is topically applied to an areaof skin in need of thickening on a subject.
 5. The method of claim 4,wherein the subject has a skin graft, an ulcer, an abrasion, an injurycaused by a repetitive impact or mechanical stress, age-related skinchanges, or skin damage due to steroid treatment.
 6. The method of claim5, wherein the ulcer is a foot ulcer.
 7. The method of claim 6, whereinthe subject has diabetes.
 8. The method of claim 5, wherein the skingraft is on a foot.
 9. The method of claim 5, wherein the area of skinin need of thickening is on a hand.
 10. The method of claim 1, whereinthe method reduces skin pigmentation.
 11. The method of claim 10,wherein the DKK1 is topically applied to a pigmented area of skin on asubject.
 12. The method of claim 11, wherein the subject has uneven skinpigmentation, hyperpigmentation, post-inflammatory pigmentation,ephelides, fragrance dermatitis, sun-damaged skin, vitiligo, a pigmentedbirthmark, lentigos, a café au lait spot, lichen simplex chronicus,melasma, porphyria cutanea tarda, Addison's disease, Peutz-Jegherssyndrome, or acanthosis nigricans.
 13. The method of claim 1, whereinthe method reduces hair growth.
 14. The method of claim 13, wherein theDKK1 is topically applied to an area of skin on a subject, whereinunwanted hair is growing on the area of skin.
 15. The method of claim14, wherein the area of skin is on an upper or lower extremity or isaxillary skin.
 16. The method of claim 14, wherein the subject hashirsutism, congenital adrenal hyperplasia, polycystic ovarian syndrome,hypertrichosis, porphyria cutanea tarda, or anorexia nervosa.
 17. Themethod of claim 1, wherein the method treats or prevents melanoma. 18.The method of claim 17, wherein the DKK1 is topically applied to an areaof skin on a subject, wherein the area of skin contains a pigmentedlesion suspected of being a pre-melanoma lesion.
 19. The method of claim1, wherein topically applying the effective amount of DKK1 tonon-palmoplantar skin comprises transdermal administration.
 20. Themethod of claim 1, wherein the DKK1 is present in a pharmaceuticalcomposition comprising an emulsion.